Enriched Graphene Oxide-Polypropylene Suture Threads Buttons Modulate the Inflammatory Pathway Induced by Escherichia coli Lipopolysaccharide.

Graphene oxide (GO), derived from graphene, has remarkable chemical-physical properties such as stability, strength, and thermal or electric conductivity and additionally shows antibacterial and anti-inflammatory properties. The present study aimed to evaluate the anti-inflammatory effects of polypropylene suture threads buttons (PPSTBs), enriched with two different concentrations of GO, in the modulation of the inflammatory pathway TLR4/MyD 88/NFκB p65/NLRP3 induced by the Escherichia coli (E. coli) lipopolysaccharide (LPS-E). The gene and the protein expression of inflammatory markers were evaluated in an in vitro model of primary human gingival fibroblasts (hGFs) by real-time PCR, western blotting, and immunofluorescence analysis. Both GO concentrations used in the polypropylene suture threads buttons-GO constructs (PPSTBs-GO) decreased the expression of inflammatory markers in hGFs treated with LPS-E. The hGFs morphology and adhesion on the PPSTBs-GO constructs were also visualized by inverted light microscopy, scanning electron microscopy (SEM), and real-time PCR. Together, these results suggest that enriched PPSTBs-GO modulates the inflammatory process through TLR4/MyD 88/NFκB p65/NLRP3 pathway.

Exploring the Assembly of Resorc[4]arenes for the Construction of Supramolecular Nano-Aggregates.

Many biologically active compounds feature low solubility in aqueous media and, thus, poor bioavailability. The formation of the host-guest complex by using calixarene-based macrocycles (i.e., resorcinol-derived cyclic oligomers) with a good solubility profile can improve solubilization of hydrophobic drugs. Herein, we explore the ability of resorc[4]arenes to self-assemble in polar solutions, to form supramolecular aggregates, and to promote water-solubility of an isoflavone endowed with anti-cancer activity, namely Glabrescione B (GlaB). Accordingly, we synthesized several architectures featuring a different pattern of substitution on the upper rim including functional groups able to undergo acid dissociation (i.e., carboxyl and hydroxyl groups). The aggregation phenomenon of the amphiphilic resorc[4]arenes has been investigated in a THF/water solution by UV-visible spectroscopy, at different pH values. Based on their ionization properties, we demonstrated that the supramolecular assembly of resorc[4]arene-based systems can be modulated at given pH values, and thus promoting the solubility of GlaB.

Material characterization and Streptococcus oralis adhesion on Polyetheretherketone (PEEK) and titanium surfaces used in implantology.

The aim of this study was to evaluate the interaction between Streptococcus oralis and Polyetheretherketone (PEEK), a novel material recently introduced in implantology. The topographical characterization and the Streptococcus oralis adhesion on this material were compared with other titanium surfaces, currently used for the production of dental implants: machined and double etched (DAE). The superficial micro-roughness of the PEEK discs was analyzed by scanning electron microscopy (SEM) and, the Energy Dispersive Spectrometer (EDS) analyzed their chemical composition. Atomic Force Microscopy (AFM) was used to characterize the micro-topography and the sessile method to evaluate the wettability of the samples. Microbiological analysis measured the colony forming units (CFUs), the biomass (OD570 detection) and the cell viability after 24 and 48 h after Streptococcus oralis cultivation on the different discs, that were previously incubated with saliva. Results showed that PEEK was characterized by a micro-roughness that was similar to machined titanium but at nano-level the nano-roughness was significantly higher in respect to the other samples. The EDS showed that PEEK superficial composition was characterized mainly by Carbonium and Oxygen. The hydrophilicity and wetting properties of PEEK were similar to machined titanium; on the contrary, double etched discs (DAE) samples were characterized by significantly higher levels (p < 0.05). PEEK was characterized by significant lower CFUs, biomass and viable cells in respect to the titanium surfaces. No differences were found between machined and DAE. The anti-adhesive and antibacterial properties showed by PEEK at 24 and 48 h against a pioneer such as S. oralis, could have an important role in the prevention of all pathologies connected with biofilm formation, like peri-implantitis in dentistry or prosthetic failures in orthopedics.

Effect of the Incorporation of Functionalized Cyclodextrins in the Liposomal Bilayer.

Liposomes loaded with drug­cyclodextrin complexes are widely used as drug delivery systems, especially for species with low aqueous solubility and stability. Investigation of the intimate interactions of macrocycles with liposomes are essential for formulation of efficient and stable drug-in-cyclodextrin-in-liposome carriers. In this work, we reported the preparation of unilamellar vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) embedded with native ß-cyclodextrin and two synthetic derivatives: heptakis(2,3,6-tri-O-methyl)-ß-cyclodextrin (TMCD) and heptakis(2,3-di-O-acetyl)-ß-cyclodextrin (DACD). We then studied the effect of these macrocycles on the liposomal size, membrane viscosity, and liposomal stability at different temperatures and concentrations. We observed that TMCD and DACD affected vesicle size and the change of size was related to CD concentration. Irrespective of its nature, the macrocycle established interactions with the phospholipidic head groups, preventing cyclodextrins to diffuse into the lipid bilayer, as confirmed by molecular dynamics simulations. Such supramolecular structuring improves liposome stability making these colloid systems promising carriers for biologically active compounds.

Fine-tuning of membrane permeability by reversible photoisomerization of aryl-azo derivatives of thymol embedded in lipid nanoparticles.

Responsiveness of liposomes to external stimuli, such as light, should allow a precise spatial and temporal control of release of therapeutic agents or ion transmembrane transport. Here, some aryl-azo derivatives of thymol are synthesized and embedded into liposomes from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine to obtain light-sensitive membranes whose photo-responsiveness, release behaviour, and permeability towards Cl- ions are investigated. The hybrid systems are in-depth characterized by dynamic light scattering, atomic force microscopy and Raman spectroscopy. In liposomal bilayer the selected guests undergo reversible photoinduced isomerization upon irradiation with UV and visible light, alternately. Non-irradiated hybrid liposomes retain entrapped 5(6)-carboxyfluorescein (CF), slowing its spontaneous leakage, whereas UV-irradiation promotes CF release, due to guest trans-to-cis isomerization. Photoisomerization also influences membrane permeability towards Cl- ions. Data processing, according to first-order kinetics, demonstrates that Cl- transmembrane transport is enhanced by switching the guest from trans to cis but restored by back-switching the guest from cis to trans upon illumination with blue light. Finally, the passage of Cl- ions across the bilayer can be fine-tuned by irradiation with light of longer λ and different light-exposure times. Fine-tuning the photo-induced structural response of the liposomal membrane upon isomerization is a promising step towards effective photo-dynamic therapy.

Exploring the Immunomodulatory Potential of Pancreatic Cancer-Derived Extracellular Vesicles through Proteomic and Functional Analyses.

Pancreatic cancer (PC) has a poor prognosis and displays resistance to immunotherapy. A better understanding of tumor-derived extracellular vesicle (EV) effects on immune responses might contribute to improved immunotherapy. EVs derived from Capan-2 and BxPC-3 PC cells isolated by ultracentrifugation were characterized by atomic force microscopy, Western blot (WB), nanoparticle tracking analysis, and label-free proteomics. Fresh PBMCs from healthy donors were treated with PC- or control-derived heterologous EVs, followed by flow cytometry analysis of CD8+ and CD4+ lymphocytes. The proteomics of lymphocytes sorted from EV-treated or untreated PBMCs was performed, and the IFN-γ concentration was measured by ELISA. Notably, most of the proteins identified in Capan-2 and BxPC-3 EVs by the proteomic analysis were connected in a single functional network (p = 1 × 10-16) and were involved in the "Immune System" (FDR: 1.10 × 10-24 and 3.69 × 10-19, respectively). Interestingly, the treatment of healthy donor-derived PBMCs with Capan-2 EVs but not with BxPC-3 EVs or heterologous control EVs induced early activation of CD8+ and CD4+ lymphocytes. The proteomics of lymphocytes sorted from EV-treated PBMCs was consistent with their activation by Capan-2 EVs, indicating IFN-γ among the major upstream regulators, as confirmed by ELISA. The proteomic and functional analyses indicate that PC-EVs have pleiotropic effects, and some may activate early immune responses, which might be relevant for the development of highly needed immunotherapeutic strategies in this immune-cold tumor.

Nutritional physiology and body composition changes during a rapid ascent to high altitude.

Exposure to high altitude might cause the body to adapt with negative energy and fluid balance that compromise body composition and physical performance. In this field study involving 12 healthy adults, sex-balanced, and aged 29 ± 4 years with a body mass index of 21.6 ± 1.8 kg/m2, we investigated the effects of a 4-day trekking up to 4556 m a.s.l. on Monte Rosa (Alps, Italy). The food intake was recorded using food diaries and nutrient averages were calculated. The bio-impedance analysis was performed at low and high altitudes, and a wearable biosensor (Swemax) was used to track hydro-saline losses in two participants. Daily total energy intake was 3348 ± 386 kcal for males and 2804 ± 415 kcal for females (13%-14% protein, 35% fat, 44%-46% carbohydrates). Although there was a significant body weight loss (65.0 ± 9.3 vs. 64.2 ± 9.10 kg, p < 0.001, d = 1.398), no significant changes in body composition parameter were found but a trend in the increase of the bioelectrical phase angle in males (p = 0.059, d = -0.991). Body water percentage significantly changed (p = 0.026, η2 p = 0.440), but the absolute water did not, suggesting that the weight loss was not due to water loss. Salivary and urinary osmolality did not change. A reduction in sweat rate at higher altitudes was observed in both participants. Interestingly, salivary leptin increased (p = 0.014, η2 p = 0.510), and salivary ghrelin decreased (p = 0.036, η2 p  = 0.403). Therefore, the 4-day trekking at altitude of hypoxia exposure induced changes in satiety and appetite hormones. High altitude expeditions require more specific nutritional guidance, and using multiplex analysis could help in monitoring fluid balance and body composition.

3D Graphene Oxide-Polyethylenimine Scaffolds for Cardiac Tissue Engineering.

The development of novel three-dimensional (3D) nanomaterials combining high biocompatibility, precise mechanical characteristics, electrical conductivity, and controlled pore size to enable cell and nutrient permeation is highly sought after for cardiac tissue engineering applications including repair of damaged heart tissues following myocardial infarction and heart failure. Such unique characteristics can collectively be found in hybrid, highly porous tridimensional scaffolds based on chemically functionalized graphene oxide (GO). By exploiting the rich reactivity of the GO's basal epoxydic and edge carboxylate moieties when interacting, respectively, with NH2 and NH3+ groups of linear polyethylenimines (PEIs), 3D architectures with variable thickness and porosity can be manufactured, making use of the layer-by-layer technique through the subsequent dipping in GO and PEI aqueous solutions, thereby attaining enhanced compositional and structural control. The elasticity modulus of the hybrid material is found to depend on scaffold's thickness, with the lowest value of 13 GPa obtained in samples containing the highest number of alternating layers. Thanks to the amino-rich composition of the hybrid and the established biocompatibility of GO, the scaffolds do not exhibit cytotoxicity; they promote cardiac muscle HL-1 cell adhesion and growth without interfering with the cell morphology and increasing cardiac markers such as Connexin-43 and Nkx 2.5. Our novel strategy for scaffold preparation thus overcomes the drawbacks associated with the limited processability of pristine graphene and low GO conductivity, and it enables the production of biocompatible 3D GO scaffolds covalently functionalized with amino-based spacers, which is advantageous for cardiac tissue engineering applications. In particular, they displayed a significant increase in the number of gap junctions compared to HL-1 cultured on CTRL substrates, which render them key components for repairing damaged heart tissues as well as being used for 3D in vitro cardiac modeling investigations.

Graphene oxide accelerates TGFß-mediated epithelial-mesenchymal transition and stimulates pro-inflammatory immune response in amniotic epithelial cells.

The application of biomaterials on immune regenerative strategies to deal with unsolved pathologies is getting attention in the field of tissue engineering. In this context, graphene oxide (GO) has been proposed as an immune-mimetic material largely used for developing stem cell-based regenerative therapies, since it has shown to influence stem cell behavior and modulate their immune response. Similarly, amniotic epithelial stem cells (AECs) are getting an increasing clinical interest as source of stem cells due to their great plasticity and immunomodulatory paracrine activities, even though GO bio-mimetic effects still remain unknown. To this aim, GO-functionalized glass coverslips have been used for AECs culture. The results demonstrated how GO-coating is able to induce and accelerate the Epithelial-Mesenchymal Transition (EMT), in a process mediated by the intracellular activation of TGFß1-SMAD2/3 signaling pathway. The trans-differentiation towards mesenchymal phenotype provides AECs of migratory ability and substantially changes the pattern of cytokines secretion upon inflammatory stimulus. Indeed, GO-exposed AECs enhance their pro-inflammatory interleukins production thus inducing a more efficient activation of macrophages and, at the same time, by slightly reducing their inhibitory action on peripheral blood mononuclear cells proliferation. Therefore, the adhesion of AECs on GO-functionalized surfaces might contribute to the generation of a tailored microenvironment useful to face both the phases of the inflammation, thereby fostering the regenerative process.

Biocompatibility and antibiofilm activity of graphene-oxide functionalized titanium discs and collagen membranes.

The aims of the study were: 1) to evaluate the effect on biofilm formation of barrier membranes and titanium surfaces coated with graphene-oxide (GO); 2) to analyze the connection between the superficial topography of the tested materials and the amount of bacterial accumulation on them and 3) to analyze the biocompatibility of GO functionalized discs using the zebrafish model. METHODS: Single species bacterial biofilms (Streptococcus oralis, Veilonella parvula, Fusobacterium nucleatum, Porphyomonas gingivalis) were grown on GO-free membranes, membranes coated with 2 and 10 µg/ml of GO, GO-free and GO-coated titanium discs. The biofilms were analyzed by determining the CFU count and by Scanning Electron Microscopy (SEM) and the materials' topography by Atomic Force Microscopy (AFM). Zebrafish model was used to determine the materials' toxicity and inflammatory effects. RESULTS: AFM showed similar roughness of control and GO-coated materials. CFU counts on GO-coated discs were significantly lower than on control discs for all species. CFU counts of S. oralis, V. parvula and P. gingivalis were lower on biofilms grown on both types of GO-coated membranes than on GO-free membrane. SEM analysis showed different formation of single species biofilm of S. oralis on control and GO-coated materials. GO-functionalized titanium discs do not induce toxic or inflammatory effects. SIGNIFICANCE: Titanium implant surfaces functionalized with GO have shown to be biocompatible and less susceptible to biofilm formation. These results encourage further in vivo investigation of the tested materials on infection prevention, specifically in prevention and reduction of peri-implant mucositis and periimplantitis incidence.

Streptococcus oralis Biofilm Formation on Titanium Surfaces.

PURPOSE: The aim of this study was to compare the Streptococcus oralis biofilm formation on titanium machined turned surfaces and sandblasted surfaces that were previously characterized for their superficial topographies. MATERIALS AND METHODS: Two commercially pure titanium surfaces were analyzed and compared: machined (turned surfaces subjected to a process of decontamination that also included a double acid attack) and sandblasted (sandblasted surfaces, cleaned with purified water, enzymatic detergent, acetone, and alcohol). The characterization of the samples at the nanolevel was performed using atomic force microscopy, which permitted calculation of the superficial nanoroughness (Ra). The sessile drop method was used to measure the water contact angle in both groups and allowed information to be gained about their wetting properties. Scanning electron microscope and energy-dispersive x-ray spectroscopy analysis allowed comparison of the microtopographic geometry and the chemical composition of the samples. Then, the disks were pre-incubated with saliva in order to form an acquired pellicle. Streptococcus oralis was put on the disks, and both groups were tested at 24 and 48 hours for biofilm biomass evaluation, colony-forming units (CFUs), and live/dead staining for cell viability. RESULTS: The sandblasted samples were characterized by a significantly higher level of superficial oxides, superficial roughness, and hydrophilicity, compared with the machined turned samples. Although there were topographic differences, the Streptococcus oralis biofilm formation, quantified in CFUs, and biomass formation at 24 and 48 hours were similar in both groups. With the live/dead staining, the sandblasted disks were characterized by an increased percentage of dead cells compared with the machined disks. CONCLUSION: Although significant topographic differences were present between machined and sandblasted disks, the Streptococcus oralis biofilm formation seems to not be significantly affected.

Calixarene-based artificial ionophores for chloride transport across natural liposomal bilayer: Synthesis, structure-function relationships, and computational study.

An amphiphilic calix[6]arene, alone or complexed with an axle to form a pseudo-rotaxane, has been embedded into liposomes prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the permeability of the membrane-doped liposomes towards Cl- ions has been evaluated by using lucigenin as the fluorescent probe. The pseudo-rotaxane promotes transmembrane transport of Cl- ions more than calix[6]arene does. Surprisingly, the quenching of lucigenin was very fast for liposomes doped with the positively charged axle alone. Molecular dynamics (MD) simulations and quantum-chemical calculations were also carried out for providing a semi-quantitative support to the experimental results.

Graphene Oxide affects Staphylococcus aureus and Pseudomonas aeruginosa dual species biofilm in Lubbock Chronic Wound Biofilm model.

Chronic wound management becomes a complex procedure because of the persistence of forming biofilm pathogens that do not respond to antimicrobial treatment. The aim of this paper is to detect the Graphene Oxide-GO effect on Staphylococcus aureus and Pseudomonas aeruginosa dual species wound biofilm in Lubbock Chronic Wound Biofilm-LCWB model. LCWB is a recognized model that mimics the spatial microbial colonization into chronic wounds and reproduces the wound and its clot. Staphylococcus aureus PECHA 10 and P. aeruginosa PECHA 4, are the pathogens used in the study. The GO effect on both in forming and mature biofilms, is detected by the evaluation of the CFU/mg reduction, the cell viability and ultrastructural analysis of the treated LCWBs. Graphene Oxide, at 50 mg/l, shows a significant antibiofilm effect in forming and mature LCWBs. In particular, during the biofilm formation, GO reduces the S. aureus and P. aeruginosa growth of 55.05% ± 4.73 and 44.18% ± 3.91 compared to the control. In mature biofilm, GO affects S. aureus and P. aeruginosa by reducing their growth of 70.24% ± 4.47 and 63.68% ± 17.56, respectively. Images taken by SEM show that GO display a disaggregated microbial effect also disrupting the fibrin network of the wound-like biofilm framework. In conclusion, GO used against microorganisms grown in LCWB, displays a significant inhibitory action resulting in a promising tool for potential application in wound management.

Liposomes Loaded with the Proteasome Inhibitor Z-Leucinyl-Leucinyl-Norleucinal are Effective in Inducing Apoptosis in Colorectal Cancer Cell Lines.

Colorectal cancer (CRC) is one of the main causes of cancer-related death in developed countries. Targeted therapies and conventional chemotherapeutics have been developed to help treat this type of aggressive cancer. Among these, the monoclonal antibodies cetuximab (Cxm) and panitumumab specifically target and inactivate the signaling of ERBB1 (EGF receptor), a key player in the development and progression of this cancer. Unfortunately, these antibodies are effective only on a small fraction of patients due to primary or secondary/acquired resistance. However, as ERBB1 cell surface expression is often maintained in resistant tumors, ERBB1 can be exploited as a target to deliver other drugs. Liposomes and immunoliposomes are under intensive investigation as pharmaceutical nanocarriers and can be functionalized with specific antibodies. In this study, we first investigated the anti-cancer activity of a cell permeable tripeptide, leucine-leucin-norleucinal (LLNle), an inhibitor of gamma-secretase and proteasome, in three different CRC cell lines that express ERBB1. We formulated LLNle-liposomes and Cxm-conjugated LLNle-loaded liposomes (LLNle-immunoliposomes) and evaluated their efficacy in inhibiting cell survival. Despite similar pro-apoptotic effects of free LLNle and LLNle-liposomes, immunoliposomes-LLNle were significantly less effective than their unconjugated counterparts. Indeed, immunoliposomes-LLNle were readily internalized and trafficked to lysosomes, where LLNle was likely trapped and/or inactivated. In conclusion, we demonstrated that LLNle was readily delivered to CRC cell lines by liposomes, but immunoliposomes-LLNle failed to show significant anti-cancer activity.

Osteoblastic Differentiation on Graphene Oxide-Functionalized Titanium Surfaces: An In Vitro Study.

BACKGROUND: Titanium implant surfaces are continuously modified to improve biocompatibility and to promote osteointegration. Graphene oxide (GO) has been successfully used to ameliorate biomaterial performances, in terms of implant integration with host tissue. The aim of this study is to evaluate the Dental Pulp Stem Cells (DPSCs) viability, cytotoxic response, and osteogenic differentiation capability in the presence of GO-coated titanium surfaces. METHODS: Two titanium discs types, machined (control, Crtl) and sandblasted and acid-etched (test, Test) discs, were covalently functionalized with GO. The ability of the GO-functionalized substrates to allow the proliferation and differentiation of DPSCs, as well as their cytotoxic potential, were assessed. RESULTS: The functionalization procedures provide a homogeneous coating with GO of the titanium surface in both control and test substrates, with unchanged surface roughness with respect to the untreated surfaces. All samples show the deposition of extracellular matrix, more pronounced in the test and GO-functionalized test discs. GO-functionalized test samples evidenced a significant viability, with no cytotoxic response and a remarkable early stage proliferation of DPSCs cells, followed by their successful differentiation into osteoblasts. CONCLUSIONS: The described protocol of GO-functionalization provides a novel not cytotoxic biomaterial that is able to stimulate cell viability and that better and more quickly induces osteogenic differentiation with respect to simple titanium discs. Our findings pave the way to exploit this GO-functionalization protocol for the production of novel dental implant materials that display improved integration with the host tissue.

Graphene Oxide Improves in vitro Fertilization in Mice With No Impact on Embryo Development and Preserves the Membrane Microdomains Architecture.

During the latest years, human infertility worsened all over the world and is nowadays reputed as a global public health issue. As a consequence, the adoption of Assisted Reproductive Technologies (ARTs) such as In Vitro Fertilization (IVF) is undergoing an impressive increase. In this context, one of the most promising strategies is the innovative adoption of extra-physiological materials for advanced sperm preparation methods. Here, by using a murine model, the addition of Graphene Oxide (GO) at a specific concentration has demonstrated to increase the spermatozoa fertilizing ability in an IVF assay, finding that 0.5 µg/ml GO addition to sperm suspensions before IVF is able to increase both the number of fertilized oocytes and embryos created with a healthy offspring given by Embryo Transplantation (ET). In addition, GO treatment has been found more effective than that carried out with methyl-ß-cyclodextrin, which represents the gold standard in promoting in vitro fertility of mice spermatozoa. Subsequent biochemical characterization of its interaction with male gametes has been additionally performed. As a result, it was found that GO exerts its positive effect by extracting cholesterol from membranes, without affecting the integrity of microdomains and thus preserving the sperm functions. In conclusion, GO improves IVF outcomes in vitro and in vivo, defining new perspectives for innovative strategies in the treatment of human infertility.