Stable hydrogel adhesion to polydimethylsiloxane enables cyclic mechanical stimulation of 3D-bioprinted smooth muscle constructs.

During normal urination, smooth muscle cells (SMCs) in the lower urinary tract (LUT) are exposed to mechanical signals that have a critical impact on tissue structure and function. Nevertheless, the mechanisms underlying the maintenance of the contractile phenotype of SMCs remain poorly understood. This is due, in part, to a lack of studies that have examined the effects of mechanical loading using three-dimensional (3D) models. In this study, surface modifications of polydimethylsiloxane (PDMS) membrane were evaluated to investigate the effects of cyclic mechanical stimulation on SMC maturation in 3D constructs. Commercially available cell stretching plates were modified with amino or methacrylate groups to promote adhesion of 3D constructs fabricated by bioprinting. After 6 days of stimulation, the effects of mechanical stimulation on the expression of contractile markers at the mRNA and protein levels were analyzed. Methacrylate-modified surfaces supported stable adhesion of the 3D constructs to the membrane and facilitated cyclic mechanical stimulation, which significantly increased the expression of contractile markers at the mRNA and protein levels. These effects were found to be mediated by activation of the p38 MAPK pathway, as inhibition of this pathway abolished the effects of stimulation in a dose-dependent manner. These results provide valuable insights into the role of mechanical signaling in maintaining the contractile phenotype of bladder SMCs, which has important implications for the development of future treatments for LUT diseases.

A surface plasmon resonance assay for characterisation and epitope mapping of anti-GLP-1 antibodies.

The incretin hormone glucagon-like peptide-1 (GLP-1) has been subject to substantial pharmaceutical research regarding the treatment of type 2 diabetes mellitus. However, quantification of GLP-1 levels remains complicated due to the low circulation concentration and concurrent existence of numerous metabolites, homologous peptides, and potentially introduced GLP-1 receptor agonists. Surface plasmon resonance (SPR) facilitates real-time monitoring allowing a more detailed characterisation of the interaction compared with conventional enzyme-linked immunosorbent assays (ELISA). In this paper, we describe the development of the first SPR assays for characterisation of anti-GLP-1 antibodies for ELISA purposes. Binding responses were obtained on covalently immobilised anti-GLP-1 antibodies at 12°C, 25°C, and 40°C and fitted to a biomolecular (1:1) interaction model showing association rates of 1.01 × 103 to 4.54 × 103  M-1  s-1 and dissociation rates of 3.56 × 10-5 to 1.56 × 10-3  s-1 leading to affinities of 35.2 to 344 nM, depending on the temperature. Determination of thermodynamic properties revealed an enthalpy driven interaction (ΔH < ΔS < 0) with higher affinities at lower temperatures due to the formation and stabilisation of hydrogen bonds within the binding site primarily composed of polar amino acids (ΔCp  < 0). Pair-wise epitope mapping was performed on captured anti-GLP-1 antibodies followed by subsequent interaction with GLP-1 (7-36) and other anti-GLP-1 antibodies. A global evaluation of every binding response led to an epitope map elucidating the potential of various anti-GLP-1 antibody pairs for sandwich ELISA and hence pinpointing the optimal antibody combinations. The SPR assays proved capable of providing vital information for ELISA development endorsing it as a useful optimisation tool.

Resistance to protein adsorption and adhesion of fibroblasts on nanocrystalline diamond films: the role of topography and boron doping.

Boron-doped nanocrystalline diamond (BNCD) films exhibit outstanding electrochemical properties that make them very attractive for the fabrication of electrodes for novel neural interfaces and prosthetics. In these devices, the physicochemical properties of the electrode materials are critical to ensure an efficient long-term performance. The aim of this study was to investigate the relative contribution of topography and doping to the biological performance of BNCD films. For this purpose, undoped and boron-doped NCD films were deposited on low roughness (LR) and high roughness (HR) substrates, which were studied in vitro by means of protein adsorption and fibroblast growth assays. Our results show that BNCD films significantly reduce the adsorption of serum proteins, mostly on the LR substrates. As compared to fibroblasts cultured on LR BNCD films, cells grown on the HR BNCD films showed significantly reduced adhesion and lower growth rates. The mean length of fibronectin fibrils deposited by the cells was significantly increased in the BNCD coated substrates, mainly in the LR surfaces. Overall, the largest influence on protein adsorption, cell adhesion, proliferation, and fibronectin deposition was due to the underlying sub-micron topography, with little or no influence of boron doping. In perspective, BNCD films displaying surface roughness in the submicron range may be used as a strategy to reduce the fibroblast growth on the surface of neural electrodes.

Aligned deposition and electrical measurements on single DNA molecules.

A reliable method of deposition of aligned individual dsDNA molecules on mica, silicon, and micro/nanofabricated circuits is presented. Complexes of biotinylated double stranded poly(dG)-poly(dC) DNA with avidin were prepared and deposited on mica and silicon surfaces in the absence of Mg(2+) ions. Due to its positive charge, the avidin attached to one end of the DNA anchors the complex to negatively charged substrates. Subsequent drying with a directional gas flow yields DNA molecules perfectly aligned on the surface. In the avidin-DNA complex only the avidin moiety is strongly and irreversibly bound to the surface, while the DNA counterpart interacts with the substrates much more weakly and can be lifted from the surface and realigned in any direction. Using this technique, avidin-DNA complexes were deposited across platinum electrodes on a silicon substrate. Electrical measurements on the deposited DNA molecules revealed linear IV-characteristics and exponential dependence on relative humidity.

Water condensation: a multiscale phenomenon.

The condensation of water is a phenomenon occurring in multiple situations in everyday life, e.g., when fog is formed or when dew forms on the grass or on windows. This means that this phenomenon plays an important role within the different fields of science including meteorology, building physics, and chemistry. In this review we address condensation models and simulations with the main focus on heterogeneous condensation of water. The condensation process is, at first, described from a thermodynamic viewpoint where the nucleation step is described by the classical nucleation theory. Further, we address the shortcomings of the thermodynamic theory in describing the nucleation and emphasize the importance of nanoscale effects. This leads to the description of condensation from a molecular viewpoint. Also presented is how the nucleation can be simulated by use of molecular models, and how the condensation process is simulated on the macroscale using computational fluid dynamics. Finally, examples of hybrid models combining molecular and macroscale models for the simulation of condensation on a surface are presented.

Engineering Photo-Cross-Linkable MXene-Based Hydrogels: Durable Conductive Biomaterials for Electroactive Tissues and Interfaces.

Light-cured conductive hydrogels have attracted immense interest in the regeneration of electroactive tissues and bioelectronic interfaces. Despite the unique properties of MXene (MX), its light-blocking effect in the range of 300-600 nm hinders the efficient cross-linking of photocurable hydrogels. In this study, we investigated the photo-cross-linking process of MX-gelatin methacrylate (GelMa) composites with different types of photoinitiators and MX concentrations to prepare biocompatible, injectable, conductive, and photocurable composite hydrogels. The examined photoinitiators were Eosin Y, Irgacure 2959 (Type I), and lithium phenyl-2,4,6-trimethylbenzoyl phosphinate (Type II). The light-blocking effect of MX strongly affected the thickness, pore structure, swelling ratio, degradation, and mechanical properties of the light-cured hydrogels. Uniform distribution of MX in the hydrogel matrix was achieved at concentrations up to 0.04 wt % but the film thickness and curing times varied depending on the type of photoinitiator. It was feasible to prepare thin films (0.5 mm) by employing Type I photoinitiators under a relatively long light irradiation (4-5 min) while thick films with centimeter sizes could be rapidly cured by using Type II photoinitiator (<60 s). The mechanical properties, including elastic modulus, toughness, and stress to break for the Type II hydrogels were significantly superior (up to 300%) to those of Type I hydrogels depending on the MX concentration. The swelling ratio was also remarkably higher (648-1274%). A conductivity of about 1 mS/cm was attained at 0.1 mg/mL MX for the composite hydrogel cured by the Type I photoinitiator. In vitro cytocompatibility assays determined that the hydrogels promoted cell viability, metabolic activity, and robust proliferation of C2C12 myoblasts, which indicated their potential to support muscle cell growth during myogenesis. The developed photocurable GelMa-MX hydrogels have the potential to serve as bioactive and conductive scaffolds to modulate cellular functions and for tissue-device interfacing.

Pore size dependence of diffuse light scattering from anodized aluminum solar cell backside reflectors.

The development of backside reflectors (BSRs) is crucial for the efficiency of future low cost thin-film silicon solar cells. In this work, the scattering efficiency of bare aluminum BSRs with different pore sizes and ordering of surface microstructures are investigated. The BSRs were fabricated by utilizing the process of self-ordering anodic oxidation on aluminum foils resulting in regions with an approximately hexagonally periodic surface microstructure. It was found that the total and diffuse light scattering reflectance spectra showed opposite tendencies when increasing the pore size of the microstructures. When the pore size was increased to 700 nm, more than 68% of the incident light with wavelengths from 250 nm to 800 nm was reflected by scattering. For a similar geometry, except that it had less ordering, this number was increased to around 80%. This large fraction of reflected light observed in the form of scattering is promising for the use of the considered geometries as BSRs in thin-film silicon solar cells.

Study of the tryptophan-terbium FRET pair coupled to silver nanoprisms for biosensing applications.

Plasmonic coupling between fluorophores and metal surfaces has become a focal point of optical research during the last two decades, however, the interactions of FRET couples with metal surfaces remain relatively unexplored. In this study, interactions of the tryptophan-Tb(3+) FRET pair with silver nanoprisms for potential biosensor development have been investigated. For this purpose an engineered lanthanide binding peptide (LBTtrp) containing tryptophan as the sensitizer for bound lanthanide ions (Tb(3+)) as well as a trypsin cleavage site was synthesized. The modified LBTtrp peptide contained two N-terminal cysteine residues to provide a stronger coupling to the silver nanoprisms (~6 nm high, ~50 nm wide). This study investigated the interaction between tryptophan, chelated Tb(3+) ions, and silver nanoprisms in solution using fluorescence and transient absorption spectroscopy. We have found that Tb(3+) luminescence decreases upon binding of the LBTtrp-Tb(3+) to silver nanoprisms and increases upon trypsin cleavage. The transient absorption spectroscopy measurements showed a significant decrease in the lifetime of the excited singlet state of tryptophan upon Tb(3+) chelation, while coupling to the silver nanoprisms did not show a significant effect on tryptophan. The results obtained in this work demonstrate a first proof of concept for a new sensitive optical biosensor in solution.

Amperometric Biosensor for Quantitative Measurement Using Sandwich Immunoassays.

State-of-the-art clinical detection methods typically involve standard immunoassay methods, requiring specialized equipment and trained personnel. This impedes their use in the Point-of-Care (PoC) environment, where ease of operation, portability, and cost efficiency are prioritized. Small, robust electrochemical biosensors provide a means with which to analyze biomarkers in biological fluids in PoC environments. Optimized sensing surfaces, immobilization strategies, and efficient reporter systems are key to improving biosensor detection systems. The signal transduction and general performance of electrochemical sensors are determined by surface properties that link the sensing element to the biological sample. We analyzed the surface characteristics of screen-printed and thin-film electrodes using scanning electron microscopy and atomic force microscopy. An enzyme-linked immunosorbent assay (ELISA) was adapted for use in an electrochemical sensor. The robustness and reproducibility of the developed electrochemical immunosensor were investigated by detecting Neutrophil Gelatinase-Associated Lipocalin (NGAL) in urine. The sensor showed a detection limit of 1 ng/mL, a linear range of 3.5-80 ng/mL, and a CV% of 8%. The results demonstrate that the developed platform technology is suitable for immunoassay-based sensors on either screen-printed or thin-film gold electrodes.

Tailoring Hydrogel Composition and Stiffness to Control Smooth Muscle Cell Differentiation in Bioprinted Constructs.

BACKGROUND: Reliable in vitro cellular models are needed to study the phenotypic modulation of smooth muscle cells (SMCs) in health and disease. The aim of this study was to optimize gelatin methacrylate (GelMA)/alginate hydrogels for bioprinting three-dimensional (3D) SMC constructs. METHODS: Four different hydrogel groups were prepared by mixing different concentrations (% w/v) of GelMA and alginate: G1 (5/1.5), G2 (5/3), G3 (7.5/1.5), and G4 (7.5/3). GelMA 10% was used as control (G5). A circular structure containing human bladder SMCs was fabricated by using an extrusion-based bioprinter. The effects of the mixing ratios on printability, viability, proliferation, and differentiation of the cells were investigated. RESULTS: Rheological analysis showed that the addition of alginate significantly stabilized the change in mechanical properties with temperature variations. The group with the highest GelMA and alginate concentrations (G4) exhibited the highest viscosity, resulting in better stability of the 3D construct after crosslinking. Compared to other hydrogel compositions, cells in G4 maintained high viability (> 80%), exhibited spindle-shaped morphology, and showed a significantly higher proliferation rate within an 8-day period. More importantly, G4 provided an optimal environment for the induction of a SMC contractile phenotype, as evidenced by significant changes in the expression of marker proteins and morphological parameters. CONCLUSION: Adjusting the composition of GelMA/alginate hydrogels is an effective means of controlling the SMC phenotype. These hydrogels support bioprinting of 3D models to study phenotypic smooth muscle adaptation, with the prospect of using the constructs in the study of therapies for the treatment of urethral strictures.

Epoxy Compositions with Reduced Flammability Based on DER-354 Resin and a Curing Agent Containing Aminophosphazenes Synthesized in Bulk Isophoronediamine.

A method for the synthesis of an amine-containing epoxy resin curing agent by dissolving hexakis-[(4-formyl)phenoxy]cyclotriphosphazene in an excess of isophoronediamine was developed. The curing agent was characterized via NMR and IR spectroscopy and MALDI-TOF mass spectrometry, and its rheological characteristics were studied. Compositions based on DER-354 epoxy resin and the synthesized curing agent with different amounts of phosphazene content were obtained. The rheological characteristics of these compositions were studied, followed by their curing. An improvement in several thermal (DSC), mechanical (compression, tension, and adhesion), and physicochemical (water absorption and water solubility) characteristics, as well as the fire resistance of the obtained materials modified with phosphazene, was observed, compared with unmodified samples. In particular, there was an improvement in adhesive characteristics and fire resistance. Thus, compositions based on a curing agent containing a 30% modifier were shown to fulfill the V-1 fire resistance category. The developed compositions can be processed by contact molding, winding, and resin transfer molding (RTM), and the resulting material is suitable for use in aircraft, automotive products, design applications, and home repairs.

Nanoaggregates of Biphilic Carboxyl-Containing Copolymers as Carriers for Ionically Bound Doxorubicin.

Application of nanocarriers for drug delivery brings numerous advantages, allowing both minimization of side effects common in systemic drug delivery and improvement in targeting, which has made it the focal point of nanoscience for a number of years. While most of the studies are focused on encapsulation of hydrophobic drugs, delivery of hydrophilic compounds is typically performed via covalent attachment, which often requires chemical modification of the drug and limits the release kinetics. In this paper, we report synthesis of biphilic copolymers of various compositions capable of self-assembly in water with the formation of nanoparticles and suitable for ionic binding of the common anticancer drug doxorubicin. The copolymers are synthesized by radical copolymerization of N-vinyl-2-pyrrolidone and acrylic acid using n-octadecyl-mercaptan as a chain transfer agent. With an increase of the carboxyl group's share in the chain, the role of the electrostatic stabilization factor of the nanoparticles increased as well as the ability of doxorubicin as an ion binder. A mathematical description of the kinetics of doxorubicin binding and release is given and thermodynamic functions for the equilibrium ionic binding of doxorubicin are calculated.

Synthesis of Amphiphilic Copolymers of N-Vinyl-2-pyrrolidone and Allyl Glycidyl Ether for Co-Delivery of Doxorubicin and Paclitaxel.

Co-delivery of chemotherapeutics in cancer treatment has been proven essential for overcoming multidrug resistance and improving the outcome of therapy. We report the synthesis of amphiphilic copolymers of N-vinyl-2-pyrrolidone and allyl glycidyl ether of various compositions and demonstrate that they can form nanoaggregates capable of simultaneous covalent immobilization of doxorubicin by the epoxy groups in the shell and hydrophobic-driven incorporation of paclitaxel into the core of nanoparticles. The structure of the obtained copolymers was characterized by 13C NMR, IR, and MALDI spectroscopy, as well as adsorption at the water/toluene interface. A linear increase in the number-average molecular weight of amphiphilic copolymers and a decrease in the number-average diameter of macromolecular aggregates with an increase in the ratio N-vinyl-2-pyrrolidone/allyl glycidyl ether were observed. The assembled nanocarriers were characterized by DLS. The reported novel nanocarriers can be of interest for delivery and co-delivery of a wide range of pharmacological preparations and combined therapy for cancer and other deceases.

Resolving the Conflict between Strength and Toughness in Bioactive Silica-Polymer Hybrid Materials.

Simultaneously improving the strength and toughness of materials is a major challenge. Inorganic-polymer hybrids offer the potential to combine mechanical properties of a stiff inorganic glass with a flexible organic polymer. However, the toughening mechanism at the atomic scale remains largely unknown. Based on combined experimental and molecular dynamics simulation results, we find that the deformation and fracture behavior of hybrids are governed by noncovalent intermolecular interactions between polymer and silica networks rather than the breakage of covalent bonds. We then attempt three methods to improve the balance between strength and toughness of hybrids, namely the total inorganic/organic (I/O) weight ratio, the size of silica nanoparticles, and the ratio of -C-O vs -C-C bonds in the polymer chains. Specifically, for a hybrid with matched silica size and I/O ratio, we demonstrate optimized mechanical properties in terms of strength (1.75 MPa at breakage), degree of elongation at the fracture point (31%), toughness (219 kPa), hardness (1.08 MPa), as well as Young's modulus (3.0 MPa). We also demonstrate that this hybrid material shows excellent biocompatibility and ability to support cell attachment as well as proliferation. This supports the possible application of this material as a strong yet tough bone scaffold material.

Melt Electrospinning of PET and Composite PET-Aerogel Fibers: An Experimental and Modeling Study.

Increasingly advanced applications of polymer fibers are driving the demand for new, high-performance fiber types. One way to produce polymer fibers is by electrospinning from polymer solutions and melts. Polymer melt electrospinning produces fibers with small diameters through solvent-free processing and has applications within different fields, ranging from textile and construction, to the biotech and pharmaceutical industries. Modeling of the electrospinning process has been mainly limited to simulations of geometry-dependent electric field distributions. The associated large change in viscosity upon fiber formation and elongation is a key issue governing the electrospinning process, apart from other environmental factors. This paper investigates the melt electrospinning of aerogel-containing fibers and proposes a logistic viscosity model approach with parametric ramping in a finite element method (FEM) simulation. The formation of melt electrospun fibers is studied with regard to the spinning temperature and the distance to the collector. The formation of PET-Aerogel composite fibers by pneumatic transport is demonstrated, and the critical parameter is found to be the temperature of the gas phase. The experimental results form the basis for the electrospinning model, which is shown to reproduce the trend for the fiber diameter, both for polymer as well as polymer-aerogel composites.

Mastication of polyolefins alters the microbial composition in Galleria mellonella.

Recent studies have indicated that Galleria mellonella larvae ingest polyethylene films and the degradation mechanism could inspire biotechnological exploitation for degrading plastic to eliminate global pollution from plastic waste. In this study, we tested the chemical compositions of masticated and ingested different plastic types by G. mellonella. High throughput sequencing of 16S rRNA gene was used to characterize the alteration of the microbial communities derived from salivary glands, gut contents and whole G. mellonella larvae. Our results indicated that G. mellonella is able to masticate polyethylene (PE), expanded polystyrene (EPS) and polypropylene (PP) and convert it to small particles with very large and chemically modified surfaces. The characteristics of the polymer affect the rate of damage. Formation of functional carbonyl groups on the appearance of oxidized metabolic intermediates of polyolefins in the frass samples observed. We found that the mastication of EPS, PP or PE could significantly alter the microbial composition in the gut content while it did not appear to influence the salivary glands microbial community. Representatives of Desulfovibrio vulgaris and Enterobacter grew with the PE diet while mastication of polystyrene and polypropylene increased the abundance of Enterococcus. The evaluation of bacterial communities in whole larvae confirmed the obtained result and additionally showed that the abundance of Paenibacillus, Corynebacterium and Commamonadaceae increased by Styrofoam (EPS) consumption.

Synthesis, Self-Assembly and In Vitro Cellular Uptake Kinetics of Nanosized Drug Carriers Based on Aggregates of Amphiphilic Oligomers of N-Vinyl-2-pyrrolidone.

Development of nanocarrier-based drug delivery systems is a major breakthrough in pharmacology, promising targeted delivery and reduction in drug toxicity. On the cellular level, encapsulation of a drug substantially affects the endocytic processes due to nanocarrier-membrane interaction. In this study we synthesized and characterized nanocarriers assembled from amphiphilic oligomers of N-vinyl-2-pyrrolidone with a terminal thiooctadecyl group (PVP-OD). It was found that the dissolution free energy of PVP-OD depends linearly on the molecular mass of its hydrophilic part up to M¯n = 2 × 104, leading to an exponential dependence of critical aggregation concentration (CAC) on the molar mass. A model hydrophobic compound (DiI dye) was loaded into the nanocarriers and exhibited slow release into the aqueous phase on a scale of 18 h. Cellular uptake of the loaded nanocarriers and that of free DiI were compared in vitro using glioblastoma (U87) and fibroblast (CRL2429) cells. While the uptake of both DiI/PVP-OD nanocarriers and free DiI was inhibited by dynasore, indicating a dynamin-dependent endocytic pathway as a major mechanism, a decrease in the uptake rate of free DiI was observed in the presence of wortmannin. This suggests that while macropinocytosis plays a role in the uptake of low-molecular components, this pathway might be circumvented by incorporation of DiI into nanocarriers.

Bio- and Hemo-Compatible Silk Fibroin PEGylated Nanocarriers for 5-Fluorouracil Chemotherapy in Colorectal Cancer: In Vitro Studies.

5-fluorouracil (5-FU) remains the gold standard of treatment for colorectal cancer, but its poor bioavailability and high systemic toxicity highlight the urgent need for the development of novel delivery strategies to increase the efficacy of 5-FU treatment. The present study is aimed to design and validate a PEGylated Silk Fibroin Nanocarrier (SF/PEG nanoparticles (NPs)) as an efficient 5-FU delivery system for potential intravenous administration. Using the human adenocarcinoma HT-29 cell line as an in vitro model for colorectal cancer, the cytotoxicity screening of the SF/PEG NPs showed that pristine nanocarriers were highly biocompatible, while the addition of 5-FU triggers a dramatic reduction in tumor cell viability, proliferation potential and mitochondrial integrity as well as a significant increase in nitric oxide production. Despite their high in vitro cytotoxicity, the 5-FU SF/PEG NPs were found hemocompatible as no impact on red blood cells hemolysis or the phagocytic activity of the granulocytes was observed. Exposure of HT-29 tumor cells and blood samples to 5-FU SF/PEG NPs augmented the tumor necrosis factor-α levels. Moreover, 5-FU SF/PEG NPs showed an impact on tumor cell migration and invasive potential as both of these processes were inhibited by the NP treatment.

PH-dependent self-assembly of the short Surfactant-like peptide KA6.

Self-assembly of amphiphilic peptides designed during the last ten years by different research groups lead to a large variety of 3D-structures that already found applications in e.g., for stabilization of large protein complexes, cell culturing systems etc. We present synthesis and characterization of a novel amphiphilic peptide KA6 that exhibits clear charge separation controllable by the pH of the environment. The self-assembly in this system is largely governed by electrostatic interaction, thus a change in pH will not only lead to a change in critical micellar concentration (CMC) of the peptide but also to the changes in micellar structure as revealed by atomic force microscopy (AFM) and circular dichroism (CD) study. At basic pH the micellar structure inverts exposing the opposite end of the peptide chain to the solution. This interesting phenomenon could provide basis for novel pH sensitive materials including drug delivery and controlled release systems.

Drug Delivery with Polymeric Nanocarriers-Cellular Uptake Mechanisms.

Nanocarrier-based systems hold a promise to become "Dr. Ehrlich's Magic Bullet" capable of delivering drugs, proteins and genetic materials intact to a specific location in an organism down to subcellular level. The key question, however, how a nanocarrier is internalized by cells and how its intracellular trafficking and the fate in the cell can be controlled remains yet to be answered. In this review we survey drug delivery systems based on various polymeric nanocarriers, their uptake mechanisms, as well as the experimental techniques and common pathway inhibitors applied for internalization studies. While energy-dependent endocytosis is observed as the main uptake pathway, the integrity of a drug-loaded nanocarrier upon its internalization appears to be a seldomly addressed problem that can drastically affect the uptake kinetics and toxicity of the system in vitro and in vivo.