3D topographies promote macrophage M2d-Subset differentiation.

In vitro cellular models denote a crucial part of drug discovery programs as they aid in identifying successful drug candidates based on their initial efficacy and potency. While tremendous headway has been achieved in improving 2D and 3D culture techniques, there is still a need for physiologically relevant systems that can mimic or alter cellular responses without the addition of external biochemical stimuli. A way forward to alter cellular responses is using physical cues, like 3D topographical inorganic substrates, to differentiate macrophage-like cells. Herein, protein secretion and gene expression markers for various macrophage subsets cultivated on a 3D topographical substrate are investigated. The results show that macrophages differentiate into anti-inflammatory M2-type macrophages, secreting increased IL-10 levels compared to the controls. Remarkably, these macrophage cells are differentiated into the M2d subset, making up the main component of tumour-associated macrophages (TAMs), as measured by upregulated Il-10 and Vegf mRNA. M2d subset differentiation is attributed to the topographical substrates with 3D fractal-like geometries arrayed over the surface, else primarily achieved by tumour-associated factors in vivo. From a broad perspective, this work paves the way for implementing 3D topographical inorganic surfaces for drug discovery programs, harnessing the advantages of in vitro assays without external stimulation and allowing the rapid characterisation of therapeutic modalities in physiologically relevant environments.

Intracellular Membrane Transport in Vascular Endothelial Cells.

The main component of blood and lymphatic vessels is the endothelium covering their luminal surface. It plays a significant role in many cardiovascular diseases. Tremendous progress has been made in deciphering of molecular mechanisms involved into intracellular transport. However, molecular machines are mostly characterized in vitro. It is important to adapt this knowledge to the situation existing in tissues and organs. Moreover, contradictions have accumulated within the field related to the function of endothelial cells (ECs) and their trans-endothelial pathways. This has induced necessity for the re-evaluation of several mechanisms related to the function of vascular ECs and intracellular transport and transcytosis there. Here, we analyze available data related to intracellular transport within ECs and re-examine several hypotheses about the role of different mechanisms in transcytosis across ECs. We propose a new classification of vascular endothelium and hypotheses related to the functional role of caveolae and mechanisms of lipid transport through ECs.

The Fast Track for Intestinal Tumor Cell Differentiation and In Vitro Intestinal Models by Inorganic Topographic Surfaces.

Three-dimensional (3D) complex in vitro cell systems are well suited to providing meaningful and translatable results in drug screening, toxicity measurements, and biological studies. Reliable complex gastrointestinal in vitro models as a testbed for oral drug administration and toxicity are very valuable in achieving predictive results for clinical trials and reducing animal testing. However, producing these models is time-consuming due to the lengthy differentiation of HT29 or other cells into mucus-producing goblet cells or other intestinal cell lineages. In the present work, HT29 cells were grown on an inorganic topographic surface decorated with a periodic pattern of micrometre-sized amorphous SiO2 structures for up to 35 days. HT29 cells on topographic surfaces were compared to undifferentiated HT29 in glucose-containing medium on glass or culture dish and with HT29 cells differentiated for 30 days in the presence of methotrexate (HT29-MTX). The cells were stained with Alcian blue for mucus, antibodies for mucus 2 (goblet cells), villin (enterocytes), lysozyme (Paneth cells), and FITC-labeled lectins to identify different cells, glycomic profiles, and cell features. We observed that HT29 cells on topographic surfaces showed more similarities with the differentiated HT29-MTX than with undifferentiated HT29. They formed islands of cell clusters, as observed for HT29-MTX. Already after 2 days, the first mucus secretion was shown by Alcian blue stain and FITC-wheat germ agglutinin. After 4-6 days, mucus was observed on the cell surface and in the intercellular space. The cell layer was undulated, and in 3D reconstruction, the cells showed a clear polarisation with a strong actin signal to one membrane. The lectins and the antibody-staining confirmed the heterogeneous composition of differentiated HT29 cells on topographic surfaces after 6-8 days, or after 6-8 days following MTX differentiation (30 days).

Complex Tumor Spheroid Formation and One-Step Cancer-Associated Fibroblasts Purification from Hepatocellular Carcinoma Tissue Promoted by Inorganic Surface Topography.

In vitro cell models play important roles as testbeds for toxicity studies, drug development, or as replacements in animal experiments. In particular, complex tumor models such as hepatocellular carcinoma (HCC) are needed to predict drug efficacy and facilitate translation into clinical practice. In this work, topographical features of amorphous silicon dioxide (SiO2) are fabricated and tested for cell culture of primary HCC cells and cell lines. The topographies vary from pyramids to octahedrons to structures named fractals, with increased hierarchy and organized in periodic arrays (square or Hexagonal). The pyramids were found to promote complex 2D/3D tissue formation from primary HCC cells. It was found that the 2D layer was mainly composed of cancer-associated fibroblasts (CAFs), while the 3D spheroids were composed of tumor cells enwrapped by a CAF layer. Compared with conventional protocols for 3D cultures, this novel approach mimics the 2D/3D complexity of the original tumor by invading CAFs and a microtumor. Topographies such as octahedrons and fractals exclude tumor cells and allow one-step isolation of CAFs even directly from tumor tissue of patients as the CAFs migrate into the structured substrate. Cell lines form spheroids within a short time. The presented inorganic topographical surfaces stimulate complex spheroid formation while avoiding additional biological scaffolds and allowing direct visualization on the substrate.

Polyphenols Epigallocatechin Gallate and Resveratrol, and Polyphenol-Functionalized Nanoparticles Prevent Enterovirus Infection through Clustering and Stabilization of the Viruses.

To efficiently lower virus infectivity and combat virus epidemics or pandemics, it is important to discover broadly acting antivirals. Here, we investigated two naturally occurring polyphenols, Epigallocatechin gallate (EGCG) and Resveratrol (RES), and polyphenol-functionalized nanoparticles for their antiviral efficacy. Concentrations in the low micromolar range permanently inhibited the infectivity of high doses of enteroviruses (107 PFU/mL). Sucrose gradient separation of radiolabeled viruses, dynamic light scattering, transmission electron microscopic imaging and an in-house developed real-time fluorescence assay revealed that polyphenols prevented infection mainly through clustering of the virions into very stable assemblies. Clustering and stabilization were not compromised even in dilute virus solutions or after diluting the polyphenols-clustered virions by 50-fold. In addition, the polyphenols lowered virus binding on cells. In silico docking experiments of these molecules against 2- and 3-fold symmetry axes of the capsid, using an algorithm developed for this study, discovered five binding sites for polyphenols, out of which three were novel binding sites. Our results altogether suggest that polyphenols exert their antiviral effect through binding to multiple sites on the virion surface, leading to aggregation of the virions and preventing RNA release and reducing cell surface binding.

Multi-sulfonated ligands on gold nanoparticles as virucidal antiviral for Dengue virus.

Dengue virus (DENV) causes 390 million infections per year. Infections can be asymptomatic or range from mild fever to severe haemorrhagic fever and shock syndrome. Currently, no effective antivirals or safe universal vaccine is available. In the present work we tested different gold nanoparticles (AuNP) coated with ligands ω-terminated with sugars bearing multiple sulfonate groups. We aimed to identify compounds with antiviral properties due to irreversible (virucidal) rather than reversible (virustatic) inhibition. The ligands varied in length, in number of sulfonated groups as well as their spatial orientation induced by the sugar head groups. We identified two candidates, a glucose- and a lactose-based ligand showing a low EC50 (effective concentration that inhibit 50% of the viral activity) for DENV-2 inhibition, moderate toxicity and a virucidal effect in hepatocytes with titre reduction of Median Tissue Culture Infectious Dose log10TCID50 2.5 and 3.1. Molecular docking simulations complemented the experimental findings suggesting a molecular rationale behind the binding between sulfonated head groups and DENV-2 envelope protein.

Reproducibility warning: The curious case of polyethylene glycol 6000 and spheroid cell culture.

Reproducibility of results is essential for a well-designed and conducted experiment. Several reasons may originate failure in reproducing data, such as selective reporting, low statistical power, or poor analysis. In this study, we used PEG6000 samples from different distributors and tested their capability inducing spheroid formation upon surface coating. MALDI-MS, NMR, FTIR, and Triple SEC analysis of the different PEG60000s showed nearly identical physicochemical properties different, with only minor differences in mass and hydrodynamic radius, and AFM analysis showed no significant differences in the surface coatings obtained with the available PEG6000s. Despite these similarities, just one showed a highly reproducible formation of spheroids with different cell lines, such as HT-29, HeLa, Caco2, and PANC-1. Using the peculiar PEG6000 sample and a reference PEG6000 chosen amongst the others as control, we tested the effect of the cell/PEG interaction by incubating cells in the PEG solution prior to cell plating. These experiments indicate that the spheroid formation is due to direct interaction of the polymer with the cells rather than by interaction of cells with the coated surfaces. The experiments point out that for biological entities, such as cells or tissues, even very small differences in impurities or minimal variations in the starting product can have a very strong impact on the reproducibility of data.

Validation of Hepatocellular Carcinoma Experimental Models for TGF-ß Promoting Tumor Progression.

Transforming growth factor beta (TGF-ß) is a pleiotropic cytokine with dual role in hepatocellular carcinoma (HCC). It acts as tumor-suppressor and tumor-promoter in the early and late stage respectively. TGF-ß influences the tumor-stroma cross-talk affecting the tumoral microenvironment. Therefore, inhibiting the TGF- ß mediated pathway alone and/or in combination with chemotherapeutics represents an important therapeutic option. Experimental models to dissect the role of TGF-ß in HCC tumor progression as well as the effectiveness of specific inhibitors are tricky. HCC cell lines respond to TGF-ß according to their epithelial phenotype. However, the mesenchymal and more aggressive HCC cell lines in vitro, do not develop tumors when transplanted in vivo, thus hampering the understanding of molecular pathways that dictate outcome. In addition, in this model the native immune system is abolished, therefore the contribution of inflammation in hepatocarcinogenesis is unreliable. Different strategies have been set up to engineer HCC animal models, including genetically modified mice, chemically induced HCC, or hydrodynamic techniques. Patient-derived xenograft is currently probably the most fascinating model, keeping in mind that models cannot mirror all the reality. In this context, we discuss the different available HCC mouse models including our experimental model treated with inhibitor of TGF-ß receptor Type I kinase (Galunisertib) and a potential role of exosomes in TGF-ß moderated tumor progression of HCC. Unfortunately, no positive results were obtained in our treated orthotopic model because it does not reproduce the critical tumor-stroma interactions of the HCC.

Patchy Amphiphilic Dendrimers Bind Adenovirus and Control Its Host Interactions and in Vivo Distribution.

The surface of proteins is heterogeneous with sophisticated but precise hydrophobic and hydrophilic patches, which is essential for their diverse biological functions. To emulate such distinct surface patterns on macromolecules, we used rigid spherical synthetic dendrimers (polyphenylene dendrimers) to provide controlled amphiphilic surface patches with molecular precision. We identified an optimal spatial arrangement of these patches on certain dendrimers that enabled their interaction with human adenovirus 5 (Ad5). Patchy dendrimers bound to the surface of Ad5 formed a synthetic polymer corona that greatly altered various host interactions of Ad5 as well as in vivo distribution. The dendrimer corona (1) improved the ability of Ad5-derived gene transfer vectors to transduce cells deficient for the primary Ad5 cell membrane receptor and (2) modulated the binding of Ad5 to blood coagulation factor X, one of the most critical virus-host interactions in the bloodstream. It significantly enhanced the transduction efficiency of Ad5 while also protecting it from neutralization by natural antibodies and the complement system in human whole blood. Ad5 with a synthetic dendrimer corona revealed profoundly altered in vivo distribution, improved transduction of heart, and dampened vector sequestration by liver and spleen. We propose the design of bioactive polymers that bind protein surfaces solely based on their amphiphilic surface patches and protect against a naturally occurring protein corona, which is highly attractive to improve Ad5-based in vivo gene therapy applications.

Urinary exosomal shuttle RNA: Promising cancer diagnosis biomarkers of lower urinary tract.

BACKGROUND: Prostate and bladder cancers continue to be the first and fourth most common cancers in men worldwide; thus there is an urgent need for more accurate biomarkers that can detect these types of cancer in a non-invasive way. Liquid biopsy is a new non-invasive tool for diagnosis and with a virtually unlimited supply urine is even more attractive resource since urinary exosomes have been discovered to contain RNAs that are hallmarks of cancer. It is challenging to assay those secreting lower amounts of molecules. METHODS: This review, based on articles identified through a PubMed/MEDLINE search, comprehensively summarizes state of the art approaches used in the discovery and validation of exosomal RNA biomarkers purified from the urine for lower urinary tract cancer. RESULTS: The combination of PCA3 and ERG has shown a relatively good improvement in diagnostic performance; examples of other potential biomarkers and the methods utilized in their discovery are also discussed in this review. CONCLUSIONS: Of these last markers, to date there are still few data to implement these for routine diagnosis.

FM19G11-Loaded Gold Nanoparticles Enhance the Proliferation and Self-Renewal of Ependymal Stem Progenitor Cells Derived from ALS Mice.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons. In ALS mice, neurodegeneration is associated with the proliferative restorative attempts of ependymal stem progenitor cells (epSPCs) that normally lie in a quiescent in the spinal cord. Thus, modulation of the proliferation of epSPCs may represent a potential strategy to counteract neurodegeneration. Recent studies demonstrated that FM19G11, a hypoxia-inducible factor modulator, induces epSPC self-renewal and proliferation. The aim of the study was to investigate whether FM19G11-loaded gold nanoparticles (NPs) can affect self-renewal and proliferation processes in epSPCs isolated from G93A-SOD1 mice at disease onset. We discovered elevated levels of SOX2, OCT4, AKT1, and AKT3, key genes associated with pluripotency, self-renewal, and proliferation, in G93A-SOD1 epSPCs at the transcriptional and protein levels after treatment with FM19G11-loaded NPs. We also observed an increase in the levels of the mitochondrial uncoupling protein (UCP) gene in treated cells. FM19G11-loaded NPs treatment also affected the expression of the cell cycle-related microRNA (miR)-19a, along with its target gene PTEN, in G93A-SOD1 epSPCs. Overall our findings establish the significant impact of FM19G11-loaded NPs on the cellular pathways involved in self-renewal and proliferation in G93A-SOD1 epSPCs, thus providing an impetus to the design of novel tailored approaches to delay ALS disease progression.

Distribution of superparamagnetic Au/Fe nanoparticles in an isolated guinea pig brain with an intact blood brain barrier.

Diagnosis and treatment of brain disorders, such as epilepsy, neurodegenerative diseases and tumors, would benefit from innovative approaches to deliver therapeutic or diagnostic compounds into the brain parenchyma, with either a homogeneous or a targeted localized distribution pattern. To assess the mechanistic aspect of penetration of nanoparticles (NPs) into the brain parenchyma, a complex, yet controlled and facilitated environment was used: the isolated guinea pig brain maintained in vitro by arterial perfusion. In this unique preparation the blood-brain barrier and the interactions between vascular and neuronal compartments are morphologically and functionally preserved. In this study, superparamagnetic Au/Fe nanoparticles (MUS:OT Au/Fe NPs), recently studied as a promising magnetic resonance T2 contrast agent with high cellular penetration, were arterially perfused into the in vitro isolated brain and showed high and homogeneous penetration through transcytosis into the brain parenchyma. Ultramicroscopy investigation of the in vitro isolated brain sections by TEM analysis of the electron-dense core of the MUS:OT Au/Fe NPs was conducted to understand NPs' brain penetration through the BBB after in vitro arterial perfusion and their distribution in the parenchyma. Our data suggest that MUS:OT Au/Fe NPs enter the brain utilizing a physiological route and therefore can be exploited as brain penetrating nanomaterials with potential contrast agent and theranostics capabilities.

Conformal coating by multilayer nano-encapsulation for the protection of human pancreatic islets: In-vitro and in-vivo studies.

To improve the efficiency of pancreatic islet transplantation, we performed in-vitro and in-vivo experiments with isolated human pancreatic islets coated by multi-layer nano-encapsulation using differently charged polymers [chitosan and poly(sodium styrene sulfonate)] to obtain up to 9 layers. The islet coating (thickness: 104.2 ±â€¯4.2 nm) was uniform, with ≥ 90% cell viability and well preserved beta- and alpha-cell ultrastructure. Nano-encapsulated islets maintained physiological glucose-stimulated insulin secretion by both static incubation and perifusion studies. Notably, palmitate- or cytokine-induced toxicity was significantly reduced in nano-coated islets. Xenotransplantation of nano-encapsulated islets under the kidney capsule of streptozotocin-induced C57Bl/6J diabetic mice allowed long term normal or near normal glycemia, associated with minimal infiltration of immune cell into the grafts, well preserved islet morphology and signs of re-vascularization. In summary, the multi-layer nano-encapsulation approach described in the present study provides a promising tool to effectively protect human islets both in-vitro andin-vivo conditions.

Selective Targeting of Proteins by Hybrid Polyoxometalates: Interaction Between a Bis-Biotinylated Hybrid Conjugate and Avidin.

The Keggin-type polyoxometalate [γ-SiW10O36]8- was covalently modified to obtain a bis-biotinylated conjugate able to bind avidin. Spectroscopic studies such as UV-vis, fluorimetry, circular dichroism, coupled to surface plasmon resonance technique were used to highlight the unique interplay of supramolecular interactions between the homotetrameric protein and the bis-functionalized polyanion. In particular, the dual recognition mechanism of the avidin encompasses (i) a complementary electrostatic association between the anionic surface of the polyoxotungstate and each positively charged avidin subunit and (ii) specific host-guest interactions between each biotinylated arm and a corresponding pocket on the tetramer subunits. The assembly exhibits peroxidase-like reactivity and it was used in aqueous solution for L-methionine methyl ester oxidation by H2O2. The recognition phenomenon was then exploited for the preparation of layer-by-layer films, whose structural evolution was monitored in situ by ATR-FTIR spectroscopy. Finally, cell tracking studies were performed by exploiting the specific interactions with a labeled streptavidin.

Evolution of Nanoparticle Protein Corona across the Blood-Brain Barrier.

Engineered nanoparticles offer the chance to improve drug transport and delivery through biological barriers, exploiting the possibility to leave the blood circulation and traverse the endothelial vascular bed, blood-brain barrier (BBB) included, to reach their target. It is known that nanoparticles gather molecules on their surface upon contact with biological fluids, forming the "protein corona", which can affect their fate and therapeutic/diagnostic performance, yet no information on the corona's evolution across the barrier has been gathered so far. Using a cellular model of the BBB and gold nanoparticles, we show that the composition of the corona undergoes dramatic quantitative and qualitative molecular modifications during passage from the "blood" to the "brain" side, while it is stable once beyond the BBB. Thus, we demonstrate that the nanoparticle corona dynamically and drastically evolves upon crossing the BBB and that its initial composition is not predictive of nanoparticle fate and performance once beyond the barrier at the target organ.

Frizzled-10 and cancer progression: Is it a new prognostic marker?

Frizzled (FZD) proteins, a family of Wnt receptors, are involved in carcinogenesis in different organs. One interesting FZD protein is FZD-10 highly expressed in embryogenesis but completely absent in the membrane or cytosol of healthy proliferated cells. We studied in detail the expression level and the location of Frizzled-10 protein in different cancerous tissues, such as colon, melanoma and gastric cancer and in function of different staging of the tumor and in metastases. We observed a correlation between cancer evolution and FZD-10 expression, and localization of protein during carcinogenesis. In colon, we have an increase of cytoplasmic FZD-10 expression from hyperplastic mucosa to metastatic tissues, while the amount in the nucleus decreases significantly in T3 and T4 staging tumors as well as in metastases. In melanoma and gastric cancer, we observed the opposite trend of FZD-10 protein in the cytosol but both show a decrease in the T3 and T4 stage of the tumor and in metastases. However, the decrease is less prominent in gastric cancer. Our findings indicate an important role of FZD-10 in tumor progression especially in the later stages of tumor. The nuclear expression of FZD-10 or its absence can give a new tool for tumor staging to pathologists. For target therapy, at least for colon cancer, the high presence of FZD-10 in the later stages of tumor progression and the absence in healthy tissue present a promising new approach.

pH-Mediated molecular differentiation for fluorimetric quantification of chemotherapeutic drugs in human plasma.

At present, drug dosage is based on standardised approaches that disregard pharmakokinetic differences between patients and lead to non-optimal efficacy and unnecessary side effects. In this work, we demonstrate the potential of pH-mediated fluorescence spectroscopy for therapeutic drug monitoring in complex media. We apply this principle to the simultaneous quantification of the chemotherapeutic prodrug Irinotecan and its active metabolite SN-38 from human plasma across the clinically relevant concentration range, i.e. from micromolar to nanomolar at molar ratios of up to 30 : 1.

Broad-spectrum non-toxic antiviral nanoparticles with a virucidal inhibition mechanism.

Viral infections kill millions yearly. Available antiviral drugs are virus-specific and active against a limited panel of human pathogens. There are broad-spectrum substances that prevent the first step of virus-cell interaction by mimicking heparan sulfate proteoglycans (HSPG), the highly conserved target of viral attachment ligands (VALs). The reversible binding mechanism prevents their use as a drug, because, upon dilution, the inhibition is lost. Known VALs are made of closely packed repeating units, but the aforementioned substances are able to bind only a few of them. We designed antiviral nanoparticles with long and flexible linkers mimicking HSPG, allowing for effective viral association with a binding that we simulate to be strong and multivalent to the VAL repeating units, generating forces (∼190 pN) that eventually lead to irreversible viral deformation. Virucidal assays, electron microscopy images, and molecular dynamics simulations support the proposed mechanism.  These particles show no cytotoxicity, and in vitro nanomolar irreversible activity against herpes simplex virus (HSV), human papilloma virus, respiratory syncytial virus (RSV), dengue and lenti virus. They are active ex vivo in human cervicovaginal histocultures infected by HSV-2 and in vivo in mice infected with RSV.