Mild Quantitative One Step Removal of Macrophages from Cocultures with Human Umbilical Vein Endothelial Cells Using Thermoresponsive Poly(Di(Ethylene Glycol)Methyl Ether Methacrylate) Brushes.

The authors report on a mild, label-free, and fast method for the separation of human umbilical vein endothelial cells (HUVEC), which are relevant cells, whose use is not limited to studies of endothelial dysfunction, from cocultures with macrophages to afford HUVEC in ≈100% purity. Poly(di(ethylene glycol)methyl ether methacrylate) (PDEGMA) brushes with a dry thickness of (5 ± 1) nm afford the highly effective one-step separation by selective HUVEC detachment, which is based on the brushes' thermoresponsive behavior. Below the thermal transition at 32 °C the brushes swells and desorbs attached proteins, resulting in markedly decreased cell adhesion. Specifically, HUVEC and macrophages, which are differentiated from THP-1 monocytes, are seeded and attached to PDEGMA brushes at 37°C. After decreasing the temperature to 22°C, HUVEC shows a decrease in their cell area, while the macrophages are not markedly affected by the temperature change. After mild flushing with a cell culture medium, the HUVEC can be released from the surface and reseeded again with ≈100% purity on a new surface. With this selective cell separation and removal method, it is possible to separate and thereby purify HUVEC from macrophages without the use of any releasing reagent or expensive labels, such as antibodies.

Photodynamic Eradication of Pseudomonas aeruginosa with Ru-Photosensitizers Encapsulated in Enzyme Degradable Nanocarriers.

The development of new approaches for the treatment of the increasingly antibiotic-resistant pathogen Pseudomonas aeruginosa was targeted by enhancing the effect of local antimicrobial photodynamic therapy (aPDT) using poly(ethylene glycol)-block-poly(lactic acid) (PEG114-block-PLAx) nanocarriers that were loaded with a ruthenium-based photosensitizer (PS). The action of tris(1,10-phenanthroline) ruthenium (II) bis(hexafluorophosphate) (RuPhen3) encapsulated in PEG114-block-PLAx micelles and vesicles was shown to result in an appreciable aPDT inactivation efficiency against planktonic Pseudomonas aeruginosa. In particular, the encapsulation of the PS, its release, and the efficiency of singlet oxygen (1O2) generation upon irradiation with blue light were studied spectroscopically. The antimicrobial effect was analyzed with two strains of Pseudomonas aeruginosa. Compared with PS-loaded micelles, formulations of the PS-loaded vesicles showed 10 times enhanced activity with a strong photodynamic inactivation effect of at least a 4.7 log reduction against both a Pseudomonas aeruginosa lab strain and a clinical isolate collected from the lung of a cystic fibrosis (CF) patient. This work lays the foundation for the targeted eradication of Pseudomonas aeruginosa using aPDT in various medical application areas.

Investigation of the Orientation and Assembly of Functionalized Microcubes at the Oil-Water Interface.

The dependence of the preferred orientation of polystyrene microcubes on surface hydrophobicity at the water/hexadecane interface is reported. Similar to the water/air interfaces, the microcubes were shown to reside at the water/hexadecane interface with three distinct orientations: face-up, edge-up, and vertex-up. Concomitantly, ordered aggregates with flat plate, tilted linear, and close-packed hexagonal structures were formed, driven by capillary force. With increasing the hydrophobicity of five sides of the cubes, the preferential microcube orientation at the water/hexadecane interface changed sequentially from face-up to edge-up, to vertex-up, then back to edge-up, and to face-up. This dependence of the preferential microcube orientation on surface hydrophobicity at the water/hexadecane interface differs from that observed at the water/air interface, where the preferential orientation changed only from face-up to edge-up, then to vertex-up, as surface hydrophobicity increased. In addition, preformed microcube assemblies at the water/air interface could be dynamically reconfigured by replacing the air phase with hexadecane under stirring.

On-Demand Cell Sheet Release with Low Density Peptide-Functionalized Non-LCST Polymer Brushes.

Cell sheet harvesting offers a great potential for the development of new therapies for regenerative medicine. For cells to adhere onto surfaces, proliferate, and to be released on demand, thermoresponsive polymeric coatings are generally considered to be required. Herein, an alternative approach for the cell sheet harvesting and rapid release on demand is reported, circumventing the use of thermoresponsive materials. This approach is based on the end-group biofunctionalization of non-thermoresponsive and antifouling poly(2-hydroxyethyl methacrylate) (p(HEMA)) brushes with cell-adhesive peptide motifs. While the nonfunctionalized p(HEMA) surfaces are cell-repellant, ligation of cell-signaling ligand enables extensive attachment and proliferation of NIH 3T3 fibroblasts until the formation of a confluent cell layer. Remarkably, the formed cell sheets can be released from the surfaces by gentle rinsing with cell-culture medium. The release of the cells is found to be facilitated by low surface density of cell-adhesive peptides, as confirmed by X-ray photoelectron spectroscopy. Additionally, the developed system affords possibility for repeated cell seeding, proliferation, and release on previously used substrates without any additional pretreatment steps. This new approach represents an alternative to thermally triggered cell-sheet harvesting platforms, offering possibility of capture and proliferation of various rare cell lines via appropriate selection of the cell-adhesive ligand.

Ruthenium(II) Polypyridyl Complexes for Antimicrobial Photodynamic Therapy: Prospects for Application in Cystic Fibrosis Lung Airways.

Antimicrobial photodynamic therapy (aPDT) depends on a variety of parameters notably related to the photosensitizers used, the pathogens to target and the environment to operate. In a previous study using a series of Ruthenium(II) polypyridyl ([Ru(II)]) complexes, we reported the importance of the chemical structure on both their photo-physical/physico-chemical properties and their efficacy for aPDT. By employing standard in vitro conditions, effective [Ru(II)]-mediated aPDT was demonstrated against planktonic cultures of Pseudomonas aeruginosa and Staphylococcus aureus strains notably isolated from the airways of Cystic Fibrosis (CF) patients. CF lung disease is characterized with many pathophysiological disorders that can compromise the effectiveness of antimicrobials. Taking this into account, the present study is an extension of our previous work, with the aim of further investigating [Ru(II)]-mediated aPDT under in vitro experimental settings approaching the conditions of infected airways in CF patients. Thus, we herein studied the isolated influence of a series of parameters (including increased osmotic strength, acidic pH, lower oxygen availability, artificial sputum medium and biofilm formation) on the properties of two selected [Ru(II)] complexes. Furthermore, these compounds were used to evaluate the possibility to photoinactivate P. aeruginosa while preserving an underlying epithelium of human bronchial epithelial cells. Altogether, our results provide substantial evidence for the relevance of [Ru(II)]-based aPDT in CF lung airways. Besides optimized nano-complexes, this study also highlights the various needs for translating such a challenging perspective into clinical practice.

Restoring Endogenous Repair Mechanisms to Heal Chronic Wounds with a Multifunctional Wound Dressing.

Current treatment of chronic wounds has been critically limited by various factors, including bacterial infection, biofilm formation, impaired angiogenesis, and prolonged inflammation. Addressing these challenges, we developed a multifunctional wound dressing-based three-pronged approach for accelerating wound healing. The multifunctional wound dressing, composed of nanofibers, functional nanoparticles, natural biopolymers, and selected protein and peptide, can target multiple endogenous repair mechanisms and represents a promising alternative to current wound healing products.

Ultra-high energy density supercapacitors using a nickel phosphide/nickel/titanium carbide nanocomposite capacitor electrode.

The low energy density of traditional supercapacitors has strongly restricted their applications. The utilization of novel capacitor electrodes to enhance the energy densities of supercapacitors is thus of great significance. Herein, a binder-free Ni12P5/Ni/TiC nanocomposite film is synthesized and further employed as the capacitor electrode. This nanocomposite film is grown by means of a chemical vapor deposition process, where Ni5TiO7 nanowires and a TiO2 layer are in situ converted into hierarchical interconnected three-dimensional (3D) Ni/Ni12P5 nanoparticles and a porous TiC matrix, respectively. Such a nanocomposite film exhibits an extremely high specific surface area and excellent conductivity, leading to its high capacitive performance. Remarkably, the multiple redox states of Ni species, namely two pairs of redox waves are observed in neutral aqueous solutions. At a current density of 10 mA cm-2, its specific capacitance in 1 M Na2SO4 aqueous solution is as high as 160.0 mF cm-2. The maximal energy density of a supercapacitor fabricated with this nanocomposite capacitor electrode is 42.6 W h kg-1 at a power density of 1550 W kg-1. Such an ultra-high energy density is even comparable with that of Li-batteries. The proposed supercapacitor thus has high potential for industrial applications.

Giant Biodegradable Poly(ethylene glycol)-block-Poly(ε-caprolactone) Polymersomes by Electroformation.

Here, the formation of giant enzyme-degradable polymersomes using the electroformation method is reported. Poly(ethylene glycol)-block-poly(ε-caprolactone) polymersomes have been shown previously to be attractive candidates for the detection of bacterial proteases and protease mediated release of encapsulated reporter dyes and antimicrobials. To maximize the efficiency, the maximization of block copolymer (BCP) vesicle size without compromising their properties is of prime importance. Thus, the physical-chemical properties of the BCP necessary to self-assemble into polymeric vesicles by electroformation are first identified. Subsequently, the morphology of the self-assembled structures is extensively characterized by different microscopy techniques. The vesicular structures are visualized for giant polymersomes by confocal laser scanning microscopy upon incorporation of reporter dyes during the self-assembly process. Using time correlated single photon counting and by analyzing the fluorescence decay curves, the nanoenvironment of the encapsulated fluorophores is unveiled. Using this approach, the hollow core structure of the polymersomes is confirmed. Finally, the encapsulation of different dyes added during the electroformation process is studied. The results underline the potential of this approach for obtaining microcapsules for subsequent triggered release of signaling fluorophores or antimicrobially active cargo molecules that can be used for bacterial infection diagnostics and/or treatment.

In Situ Study of Layer-by-Layer Polyelectrolyte Deposition in Nanopores of Anodic Aluminum Oxide by Reflectometric Interference Spectroscopy.

The modification of cylindrical anodic aluminum oxide (AAO) nanopores by alternating layer-by-layer (LBL) deposition of poly(sodium-4-styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) was studied in situ by reflectometric interference spectroscopy (RIfS). In particular, the kinetics of polyelectrolyte deposition inside the pores with a diameter of 37 ± 3 nm and a length of 3.7 ± 0.3 µm were unraveled, and potential differences in the LBL multilayer growth compared to flat silicon substrates as well as the effect of different ionic strengths and different types of ions were investigated. RIfS measures the effective optical thicknesses, which is-for a constant pore length-proportional to the effective refractive index of the AAO sample, from which, in turn, the deposited mass of the polymer or the corresponding layer thickness can be estimated. Compared to the multilayer growth by the LBL deposition on the flat aminosilane-primed silicon wafers, which was assessed by spectroscopic ellipsometry, the thickness increment per deposited bilayer, as well as the dependence of this increment on the ionic strength (0.01-0.15) and the counterion type (Na+ vs Ca2+) inside the aminosilane-primed nanopores, was for the first bilayers to within the experimental error identical. For thicker multilayers, the pore diameter became smaller, which led to reduced thickness increments and eventually virtually completely filled the pores. The observed kinetics is consistent with the mass-transport-limited adsorption of the polyelectrolyte to the charged surface according to a Langmuir isotherm with a negligible desorption rate. In addition to fundamental insights into the buildup of polyelectrolyte multilayers inside the AAO nanopores, our results highlight the sensitivity of RIfS and its use as an analytical tool for probing processes inside the nanopores and for the development of biosensors.

Anodic Aluminum Oxide Nanopore Template-Assisted Fabrication of Nanostructured Poly(vinyl alcohol) Hydrogels for Cell Studies.

The effect of systematically varied mechanical properties and nano- and microscale surface topography on the adhesion and proliferation of human pancreatic cancer cells on fibronectin-functionalized poly(vinyl alcohol) (PVA) hydrogels was studied to understand the impact of these properties of the cell microenvironment on cell attachment and spreading. The mechanical properties of PVA, as assessed by atomic force microscopy (AFM) nanoindentation, were varied by the number of freezing-thawing cycles in the physical cross-linking process used for the generation of the hydrogels. Nano- and micropatterned hydrogel surfaces exposing nanosized PVA pillars and cuboids were fabricated by replicating ordered cylindrical nanopores of anodic aluminum oxide (AAO) and poly(dimethylsiloxane) (PDMS) templates, respectively. Softer PVA hydrogels, functionalized covalently with fibronectin, showed enhanced cell adhesion and proliferation of PaTu 8988t cells in comparison to stiffer hydrogels. In addition, PaTu 8988t cells favored the nanopatterned surfaces over micropatterned and flat hydrogels.

Tailored Combinatorial Microcompartments through the Self-Organization of Microobjects: Assembly, Characterization, and Cell Studies.

A new concept enables the generation of cell microenvironments by microobject assembly at an water/air interface. As the orientation of 30 µm sized polymer cubes and their capillary force assembly are controlled by the surface wettability, which in turn can be modulated by coating the initially exposed surfaces with gold and self-assembled monolayers, unique niches in closely packed arrays of cubes with vertex up orientation can be realized. The random assembly of distinctly different cubes, prefunctionalized or surface-structured exclusively on their top surface, facilitates the parallel generation of different microenvironments in a combinatorial manner, which paves the way to future systematic structure-property relationship studies with cells.

Toward Label-Free Selective Cell Separation of Different Eukaryotic Cell Lines Using Thermoresponsive Homopolymer Layers.

Ultrathin thermoresponsive poly(di(ethylene glycol) methyl ether methacrylate) (PDEGMA) homopolymer layers are reported as a novel platform for label-free temperature-stimulated cell separation from a coculture of eukaryotic cell lines. Pancreatic tumor cells (PaTu 8988t) and fibroblasts (NIH 3T3) were shown to attach and proliferate on PDEGMA layers with a dry thickness of 5 ± 1 nm at 37 °C. After the cell medium cooled to below the lower critical solution temperature (LCST), PaTu 8988t cells showed a significantly decreased cell area. By contrast, there was no significant change in cell area for NIH 3T3 cells, while they still exhibited lamellipodia and filopodia. PaTu 8988t cells could be detached at 22 °C after flushing gently with the cell medium, while negligible cell detachment was observed for NIH 3T3 cells under identical conditions. This significant difference between PaTu 8988t and NIH 3T3 was due to the different cell adhesion on the culture-medium-derived protein covering the PDEGMA layer. Thus, cell separation from a coculture was achieved successfully by cooling the cell medium to 22 °C, resulting in the desorption of 95 ± 6% of PaTu 8988t cells, while the NIH 3T3 cells remained adherent. Our findings provide an effective, label-free, rapid, and simple approach, exploiting a thermoresponsive homopolymer layer to separate and enrich or isolate cells from mixed cell populations for basic biological research, medical diagnostics, tissue engineering, and cell therapy.

Thickness-Encoded Micropatterns in One-Component Thermoresponsive Polymer Brushes for Culture and Triggered Release of Pancreatic Tumor Cell Monolayers and Spheroids.

Fabrication, characterization, and application of micropatterned one-component poly(di(ethylene glycol)methyl ether methacrylate) (PDEGMA) brushes for monolayer cell and spheroid culture and temperature-triggered release are reported. Micropatterns of various shapes and sizes were designed to possess a unique functionality imparted by thermoresponsive thin PDEGMA patches, which are cell adhesive at 37 °C, embedded in a much thicker cell-resistant PDEGMA matrix that does not exhibit measurable thermoresponsive properties. Depending on the cell seeding density, PaTu 8988t human pancreatic tumor cells or spheroids were cultured area-selectively, confined by the 40 ± 4 nm thick passivating PDEGMA matrix, and could be released on demand by a mild thermally triggered brush swelling in the 5 ± 1 nm thin regions. As shown by surface plasmon resonance (SPR) measurements, in contrast to the thinner brushes, the thicker brushes exhibited virtually no fibronectin adhesive properties at 37 °C, whereas at 25 °C, both areas showed similar protein resistant behavior. The quasi-2D thickness-encoded micropatterns were shown to be useful templates for the growth of 3D multicellular aggregates. Thermally induced release after 5 days of incubation afforded 3D cell spheroids comprising up to 99% viable cells demonstrating that the system can be used as a 3D spheroid in vitro model for basic tumor research and anticancer drug screenings.

Fluorimetric Detection of G-Quadruplex DNA in Solution and Adsorbed on Surfaces with a Selective Trinuclear Cyanine Dye.

Quadruplex DNA, which is a relevant target for anticancer therapies, may alter its conformation because of interactions with interfaces. In pursuit of a versatile methodology to probe adsorption-induced conformational changes, the interaction between a fluorescent [2.2.2]heptamethinecyanine dye and quadruplex DNA (G4-DNA) was studied in solution and on surfaces. In solution, the cyanine dye exhibits a strong light-up effect upon the association with G4-DNA without interference from double-stranded DNA. In addition, a terminal π-stacking as a binding mode between the cyanine dye and G4-DNA is concluded using NMR spectroscopy. To unravel the effects of adsorption on the conformation of quadruplex-DNA, G4-DNA, and double-stranded and single-stranded DNA were adsorbed to positively charged poly(allylamine) hydrochloride (PAH) surfaces, both in planar and in constrained 55 nm diameter aluminum oxide nanopore formats. All DNA forms showed a very strong affinity to the PAH surfaces as shown by surface plasmon resonance and reflectometric interference spectroscopy. The significant increase of the fluorescence emission intensity of the cyanine light-up probe observed exclusively for surface immobilized G4-DNA affords evidence for the adsorption of G4-DNA on PAH with retained quadruplex conformation.

Ruthenium(II) Polypyridyl Complexes as Photosensitizers for Antibacterial Photodynamic Therapy: A Structure-Activity Study on Clinical Bacterial Strains.

As a growing public health concern, the worldwide spread of antimicrobial resistance urges the development of new therapies. Antibacterial photodynamic therapy (a-PDT) may be an alternative to conventional antibiotic therapy. Herein we report the synthesis and characterization of seven original reactive oxygen species (ROS)-producing ruthenium(II) polypyridyl complexes. These are part of a collection of 17 derivatives varying in terms of the nature of the substituent(s), molecular symmetry, electrical charge, and counterions. They were characterized by considering 1) their physical properties (absorption coefficient at irradiation wavelength, 1 O2 generation quantum yield, luminescence) and 2) their antibacterial activity in a series of photodynamic assays using Gram-positive and Gram-negative bacteria of clinical relevance. The results unveiled some structure-activity relationships: one derivative that combines multiple beneficial features for a-PDT was effective against all the bacteria considered, regardless of their Gram status, species, or antibiotic resistance profile. This systematic study could guide the design of next-generation ruthenium-based complexes for enhanced antibacterial photodynamic strategies.

Three-Dimensional Microstructured Poly(vinyl alcohol) Hydrogel Platform for the Controlled Formation of Multicellular Cell Spheroids.

Three-dimensional (3D) multicellular cell spheroids (MCSs) are excellent in vitro cell models, in which, e.g., the in vivo cell-cell interaction processes are much better mimicked than in conventional two-dimensional (2D) cell layers. However, the difficulties in the generation of well-defined MCSs with controlled size severely limit their application. Herein, low-adhesive poly(vinyl alcohol) (PVA) hydrogels structured with inverted pyramid-shaped microwells were used to guide the aggregation of cells into MCSs. The cells settling down into the microwells by gravity accumulated at the central tip of the wells and then gradually grew into spheroids. The size of cell spheroids can be straightforwardly controlled by the culture time and initially seeded cell number. The MCSs generated in a parallel microarray format were further used for drug testing. Our results suggest in agreement with complementary literature data that the cell culture format plays a critical role in the cellular response to drugs, and also confirms that spheroids possess a much higher drug resistance than cells in 2D layers. This novel microstructured PVA hydrogel is expected to offer a potential platform for the facile preparation of spheroids for various applications in the biomedical field.

Enhanced cell adhesion on a bio-inspired hierarchically structured polyester modified with gelatin-methacrylate.

Herein, fabrication and modification of novel bio-inspired microwell arrays with nanoscale topographic structures are reported. The natural nano- and microstructures present on the surface of rose petals were hypothesized to enhance cell-surface contacts. Thus hierarchically structured polyethylene terephthalate glycol modified (PETG) substrates were fabricated by replication from rose petals via nanoimprint lithography, followed by covalent modification and crosslinking with RGD-presenting gelatin-methacrylate (GelMA) for promoting cell adhesion and spreading. Cell culture experiments showed that the introduction of gelatin resulted in significantly enhanced cell adhesion and more than doubled cell areas on the GelMA modified surfaces. In addition, a slight preference was observed for concave compared to convex surfaces, which is tentatively attributed to the matching curvature of the micro-cavities and the cells, facilitating the accommodation of cells. These bioinspired hierarchically structured and gelatin functionalized substrates may provide new prospects for designing cell-based interfaces for advanced biomedical studies, e.g. for cell culture and biosensing in the future.

Detailed Analysis of Pancreatic Tumor Cell Attachment on Gradient PDEGMA Brushes.

Poly(di(ethylene glycol) methyl ether methacrylate) (PDEGMA) brushes show interesting thermoresponsive behavior that can be applied for cell release surfaces. Here it is shown that PDEGMA thickness gradients, which are synthesized by surface-initiated atom transfer radical polymerization, allow the systematic and precise analysis of the attachment of PaTu 8988 cells. By pumping the polymerization solution into the reactor with vertically fixed initiator samples, PDEGMA gradients with linearly increasing dry ellipsometric thickness with typical slopes of 2.5 nm cm-1 are obtained. A very narrow transition of PaTu 8988t cell attachment is observed that starts for a thickness larger than 7.1 ± 0.2 nm. For PDEGMA layers thicker than 8.7 ± 0.2 nm no attached cells are found. This very narrow transition in brush properties within a thickness difference of <2 nm from cell-adherent to cell-nonadherent can be determined in much greater detail than before owing to the thickness gradients with shallow slope.

κ-Carrageenan Enhances the Biomineralization and Osteogenic Differentiation of Electrospun Polyhydroxybutyrate and Polyhydroxybutyrate Valerate Fibers.

Novel electrospun materials for bone tissue engineering were obtained by blending biodegradable polyhydroxybutyrate (PHB) or polyhydroxybutyrate valerate (PHBV) with the anionic sulfated polysaccharide κ-carrageenan (κ-CG) in varying ratios. In both systems, the two components phase separated as shown by FTIR, DSC and TGA. According to the contact angle data, κ-CG was localized preferentially at the fiber surface in PHBV/κ-CG blends in contrast to PHB/κ-CG, where the biopolymer was mostly found within the fiber. In contrast to the neat polyester fibers, the blends led to the formation of much smaller apatite crystals (800 nm vs 7 µm). According to the MTT assay, NIH3T3 cells grew in higher density on the blend mats in comparison to neat polyester mats. The osteogenic differentiation potential of the fibers was determined by SaOS-2 cell culture for 2 weeks. Alizarin red-S staining suggested an improved mineralization on the blend fibers. Thus, PHBV/κ-CG fibers resulted in more pronounced bioactive and osteogenic properties, including fast apatite-forming ability and deposition of nanosized apatite crystals.

Bioinspired Hierarchically Structured Surfaces for Efficient Capture and Release of Circulating Tumor Cells.

The development of novel bioinspired surfaces with hierarchical micro- and nanoscale topographic structures for efficient capture and release of circulating tumor cells (CTCs) is reported. The capture of CTCs, facilitated by surface-immobilized epithelial cell adhesion molecule antibodies (anti-EpCAM), was shown to be significantly enhanced in novel three-dimensional hierarchically structured surfaces that were fabricated by replicating the natural micro- and nanostructures of rose petals. Under static conditions, these hierarchical capture substrates exhibited up to 6 times higher cell capture ability at concentrations of 100 cells mL-1 in contrast to flat anti-EpCAM-functionalized polydimethylsiloxane (PDMS) surfaces. As indicated by scanning electron microscopy (SEM) and immunofluorescent images, this enhancement can be in large part attributed to the topographical interaction between nanoscale cell surface components and nanostructures on the substrate. Similarly, the increased surface area affords a higher nominal coverage of anti-EpCAM, which increases the number of available binding sites for cell capture. By treating the substrates with the biocompatible reductant glutathione (GSH), up to 85% of the captured cells were released, which displayed over 98% cell viability after culturing on tissue culture polystyrene (TCP) for 24 h. Therefore, these bioinspired hierarchically structured and functionalized substrates can be successfully applied to capture CTCs, as well as release CTCs for subsequent analysis. These findings provide new prospects for designing cell-material interfaces for advanced cell-based biomedical studies in the future.