Producing thick films of conducting polymers by a low-cost manufacturing technique would enable new applications. However, removing huge solvent volume from diluted suspension or dispersion (1-3 wt%) in which conducting polymers are typically obtained is a true manufacturing challenge. In this work, a procedure is proposed to quickly remove water from the conducting polymer poly(3,4-ethylenedioxythiophene:poly(4-styrene sulfonate) (PEDOT:PSS) suspension. The PEDOT:PSS suspension is first flocculated with 1 m H2SO4 transforming PEDOT nanoparticles (approximate to 50-500 nm) into soft microparticles. A filtration process inspired by pulp dewatering in a paper machine on a wire mesh with apertures dimension between 60 mu m and 0.5 mm leads to thick free-standing films (approximate to 0.5 mm). Wire mesh clogging that hinders dewatering (known as dead-end filtration) is overcome by adding to the flocculated PEDOT: PSS dispersion carbon fibers that aggregate and form efficient water channels. Moreover, this enables fast formation of thick layers under simple atmospheric pressure filtration, thus making the process truly scalable. Thick freestanding PEDOT films thus obtained are used as electrocatalysts for efficient reduction of oxygen to hydrogen peroxide, a promising green chemical and fuel. The inhomogeneity of the films does not affect their electrochemical function.

Tumor associated macrophages (TAM) are key pathogenic factors in neoplastic diseases. They are known to have plasticity and can polarize into two opposing phenotypes, including the tumoricidal M1 and the protumoral M2 phenotypes with high prevalence of M2-phentoypes in patients with poor prognosis. Strategies for targeting M2-TAM may consequently increase the efficacy of therapeutic strategies for cancer treatment. Gold nanorod-assisted plasmonic photothermal therapy (PPTT) has emerged as a promising treatment for cancer but the effects of macrophage polarization parameters in the performance of this new treatment modality is still unknown. Herein, human monocytic THP-1 cells were polarized into two opposite phenotypic macrophages (M1-TAM and M2-TAM) and their response to PPTT was examined. M2-TAM exhibits a three-fold increase in AuNP uptake compared to M1-TAM. Laser irradiation results in selective killing of pro-tumoral M2-TAM after treatment with AuNPs with limited effects on anti-tumoral M1-TAM. A positive correlation between the expression of CD206 marker and the AuNP uptake may indicate the role of CD206 in facilitating AuNP uptake. Our findings also suggest that the differences in AuNP avidity and uptake between the M1-TAM and M2-TAM phenotypes may be the rationale behind the effectiveness of PPTT in the treatment of solid tumors.

Biomimetic chiral optoelectronic materials can be utilized in electronic devices, biosensors and artificial enzymes. Herein, this work reports the chiro-optical properties and architectural arrangement of optoelectronic materials generated from self-assembly of initially nonchiral oligothiophene-porphyrin derivatives and random coil synthetic peptides. The photo-physical- and structural properties of the materials were assessed by absorption-, fluorescence- and circular dichroism spectroscopy, as well as dynamic light scattering, scanning electron microscopy and theoretical calculations. The materials display a three-dimensional ordered helical structure and optical activity that are observed due to an induced chirality of the optoelectronic element upon interaction with the peptide. Both these properties are influenced by the chemical composition of the oligothiophene-porphyrin derivative, as well as the peptide sequence. We foresee that our findings will aid in developing self-assembled optoelectronic materials with dynamic architectonical accuracies, as well as offer the possibility to generate the next generation of materials for a variety of bioelectronic applications.

Hydrogels are used in a wide range of biomedical applications, including three-dimensional (3D) cell culture, cell therapy and bioprinting. To enable processing using advanced additive fabrication techniques and to mimic the dynamic nature of the extracellular matrix (ECM), the properties of the hydrogels must be possible to tailor and change over time with high precision. The design of hydrogels that are both structurally and functionally dynamic, while providing necessary mechanical support is challenging using conventional synthesis techniques. Here, we show a modular and 3D printable hydrogel system that combines a robust but tunable covalent bioorthogonal cross-linking strategy with specific peptide-folding mediated interactions for dynamic modulation of cross-linking and functionalization. The hyaluronan-based hydrogels were covalently cross-linked by strain-promoted alkyne-azide cycloaddition using multi-arm poly(ethylene glycol). In addition, ade novodesigned helix-loop-helix peptide was conjugated to the hyaluronan backbone to enable specific peptide-folding modulation of cross-linking density and kinetics, and hydrogel functionality. An array of complementary peptides with different functionalities was developed and used as a toolbox for supramolecular tuning of cell-hydrogel interactions and for controlling enzyme-mediated biomineralization processes. The modular peptide system enabled dynamic modifications of the properties of 3D printed structures, demonstrating a novel route for design of more sophisticated bioinks for four-dimensional bioprinting.

Hydrogels of cellulose nanofibrils (CNFs) are promising wound dressing candidates due to their biocompatibility, high water absorption, and transparency. Herein, two different commercially available wood species, softwood and hardwood, were subjected to TEMPO-mediated oxidation to proceed with delignification and oxidation in a one-pot process, and thereafter, nanofibrils were isolated using a high-pressure microfluidizer. Furthermore, transparent nanofibril hydrogel networks were prepared by vacuum filtration. Nanofibril properties and network performance correlated with oxidation were investigated and compared with commercially available TEMPO-oxidized pulp nanofibrils and their networks. Softwood nanofibril hydrogel networks exhibited the best mechanical properties, and in vitro toxicological risk assessment showed no detrimental effect for any of the studied hydrogels on human fibroblast or keratinocyte cells. This study demonstrates a straightforward processing route for direct oxidation of different wood species to obtain nanofibril hydrogels for potential use as wound dressings, with softwood having the most potential.

The use of conventional antibiotics has substantial clinical efficacy, however these vital antimicrobial agents are becoming less effective due to the dramatic increase in antibiotic-resistant bacteria. Novel approaches to combat bacterial infections are urgently needed and bacteriocins represent a promising alternative. In this study, the activities of the two-peptide bacteriocin PLNC8 alpha beta were investigated against different Staphylococcus spp. The peptide sequences of PLNC8 alpha and beta were modified, either through truncation or replacement of all L-amino acids with D-amino acids. Both L- and D-PLNC8 alpha beta caused rapid disruption of lipid membrane integrity and were effective against both susceptible and antibiotic resistant strains. The D-enantiomer was stable against proteolytic degradation by trypsin compared to the L-enantiomer. Of the truncated peptides, beta 1-22, beta 7-34 and beta 1-20 retained an inhibitory activity. The peptides diffused rapidly (2min) through the bacterial cell wall and permeabilized the cell membrane, causing swelling with a disorganized peptidoglycan layer. Interestingly, sub-MIC concentrations of PLNC8 alpha beta substantially enhanced the effects of different antibiotics in an additive or synergistic manner. This study shows that PLNC8 alpha beta is active against Staphylococcus spp. and may be developed as adjuvant in combination therapy to potentiate the effects of antibiotics and reduce their overall use.

The self-assembly of nanocellulose in the form of cellulose nanofibers (CNFs) can be accomplished via hydrogen-bonding assistance into completely bio-based hydrogels. This study aimed to use the intrinsic properties of CNFs, such as their ability to form strong networks and high absorption capacity and exploit them in the sustainable development of effective wound dressing materials. First, TEMPO-oxidized CNFs were separated directly from wood (W-CNFs) and compared with CNFs separated from wood pulp (P-CNFs). Second, two approaches were evaluated for hydrogel self-assembly from W-CNFs, where water was removed from the suspensions via evaporation through suspension casting (SC) or vacuum-assisted filtration (VF). Third, the W-CNF-VF hydrogel was compared to commercial bacterial cellulose (BC). The study demonstrates that the self-assembly via VF of nanocellulose hydrogels from wood was the most promising material as wound dressing and displayed comparable properties to that of BC and strength to that of soft tissue.

The safety of the cells used for Advanced Therapy Medicinal Products is crucial for patients. Reliable methods for the cell purification are very important for the commercialization of those new therapies. With the large production scale envisioned for commercialization, the cell isolation methods need to be efficient, robust, operationally simple and generic while ensuring cell biological functionality and safety. In this study, we used high magnetized magnetic agarose-based beads conjugated with protein A to develop a new method for cell separation. A high separation efficiency of 91% yield and consistent isolation performances were demonstrated using population mixtures of human mesenchymal stem cells and HER2(+) SKBR3 cells (80:20, 70:30 and 30:70). Additionally, high robustness against mechanical stress and minimal unspecific binding obtained with the protein A base conjugated magnetic beads were significant advantages in comparison with the same magnetic microparticles where the antibodies were covalently conjugated. This study provided insights on features of large high magnetized microparticles, which is promising for the large-scale application of cell purification.

Neutron reflectivity at the solid/liquid interface offers unique opportunities for resolving the structure–function relationships of interfacial layers in soft matter science. It is a non-destructive technique for detailed analysis of layered structures on molecular length scales, providing thickness, density, roughness, and composition of individual layers or components of adsorbed films. However, there are also some well-known limitations of this method, such as the lack of chemical information, the difficulties in determining large layer thicknesses, and the limited time resolution. We have addressed these shortcomings by designing and implementing a portable sample environment for in situ characterization at neutron reflectometry beamlines, integrating infrared spectroscopy under attenuated total reflection for determination of molecular entities and their conformation, and spectroscopic ellipsometry for rapid and independent measurement of layer thicknesses and refractive indices. The utility of this combined setup is demonstrated by two projects investigating (a) pH-dependent swelling of polyelectrolyte layers and (b) the impact of nanoparticles on lipid membranes to identify potential mechanisms of nanotoxicity. 

Control over the photophysical properties and molecular organization of pi-conjugated oligothiophenes is essential to their use in organic electronics. Herein we synthesized and characterized a variety of anionic pentameric oligothiophenes with different substitution patterns of L- or D-tyrosine at distinct positions along the thiophene backbone. Spectroscopic, microscopic, and theoretical studies of L- or D-tyrosine substituted pentameric oligothiophene conjugates revealed the formation of optically active pi-stacked self-assembled aggregates under acid conditions. The distinct photophysical characteristics, as well as the supramolecular structures of the assemblies, were highly influenced by the positioning of the L- or D-tyrosine moieties along the thiophene backbone. Overall, the obtained results clearly demonstrate how fundamental changes in the position of the enantiomeric side-chain functionalities greatly affect the optical properties as well as the architecture of the self-assembled supramolecular structures.

Background: Most high-throughput screening (HIS) systems studying the cytotoxic effect of chimeric antigen receptor (CAR) T cells on tumor cells rely on two-dimensional cell culture that does not recapitulate the tumor micro-environment (TME). Tumor spheroids, however, can recapitulate the TME and have been used for cytotoxicity assays of CART cells. But a major obstacle to the use of tumor spheroids for cytotoxicity assays is the difficulty in separating unbound CART and dead tumor cells from spheroids. Here, we present a three-dimensional hanging spheroid plate (3DHSP), which facilitates the formation of spheroids and the separation of unbound and dead cells from spheroids during cytotoxicity assays.

Results: The 3DHSP is a 24-well plate, with each well composed of a hanging dripper, spheroid wells, and waste wells. In the dripper, a tumor spheroid was formed and mixed with CART cells. In the 3DHSP, droplets containing the spheroids were deposited into the spheroid separation well, where unbound and dead T and tumor cells were separated from the spheroid through a gap into the waste well by tilting the 3DHSP by more than 20 degrees. Human epidermal growth factor receptor 2 (HER2)-positive tumor cells (BT474 and SKOV3) formed spheroids of approximately 300-350 pm in diameter after 2 days in the 3DHSP. The cytotoxic effects ofT cells engineered to express CAR recognizing HER2 (HER2-CAR T cells) on these spheroids were directly measured by optical imaging, without the use of live/dead fluorescent staining of the cells. Our results suggest that the 3DHSP could be incorporated into a HTS system to screen for CARs that enable T cells to kill spheroids formed from a specific tumor type with high efficacy or for spheroids consisting of tumor types that can be killed efficiently by T cells bearing a specific CAR.

Conclusions: The results suggest that the 3DHSP could be incorporated into a HTS system for the cytotoxic effects of CART cells on tumor spheroids.

Nanoparticles (NPs) are particles with more than one dimension between 1 and 100 nm. Because of their small size, they typically display different physical and chemical properties than the corresponding bulk materials. NPs have been used in many different applications, such as in electronics, optics, catalysis, and in biomedicine. Due to their colloidal nature, NPs are often immobilized on a solid substrate, such as glass or polymer-based materials, including biopolymers. Nanocellulose is a biopolymerbased nanomaterial that can be obtained from plants or bacterial biofilms. They can be processed into thin and highly hydrated films with high mechanical strength and can serve as a versatile substrate for NPs. Bacterial cellulose (BC) is also an interesting material for generating wound dressings. The combination of NPs and BC results in soft and flexible nanocomposites (BC-NPs) that can demonstrate novel properties and improve the functionality of wound dressings. 

BC-NP nanocomposites have previously been obtained by impregnating BC with the reactants needed for synthesis of the NPs and allowing the reaction to proceed in situ, inside and on the surface of the BC. This strategy limits the possibilities to control NP geometry and NP concentration and make synthesis of nanocomposites with more sophisticated compositions very challenging. In addition, the synthesis conditions used can potentially have negative effects on the properties of BC. 

The work presented in this thesis shows the possibility to produce well-defined, tunable BC-NP nanocomposites using self-assembly under very benign conditions that enable functionalization of BC with a wide range of different types of NPs. In addition to exploring the self-assembly process and the physical properties of these new BC-NP composites, several different applications were investigated. The functionalization of BC with gold nanoparticles (AuNPs) of different sizes and geometries was demonstrated. The resulting materials were used for development of a new sensor transduction technology, exploiting the optical response upon mechanical compression to detect biomolecules. BC-AuNP nanocomposites were also developed for monitoring of protease activity of wound pathogens, for catalysis, and for fabrication of ultra-black materials with unique absorption and scattering profiles of light in the visible and near infrared spectral range. In addition, the self-assembly process could be adopted for generating BC-mesoporous silica nanoparticles (MSNs) nanocomposite wound dressings. The resulting high surface area materials could be used as carriers for pH sensitive dyes. The pH-responsive BC-MSNs demonstrated adequate biocompatibility and allowed for monitoring of wound pH and for assessment of wound status. 

The strategies for functionalization of BC with inorganic NPs that was developed and explored in this thesis are highly versatile and allow for fabrication of a wide range of multifunctional nanocomposite materials. 

Nanocomposites of metal nanoparticles (NPs) and bacterial nanocellulose (BC) enable fabrication of soft and biocompatible materials for optical, catalytic, electronic, and biomedical applications. Current BC-NP nanocomposites are typically prepared by in situ synthesis of the NPs or electrostatic adsorption of surface functionalized NPs, which limits possibilities to control and tune NP size, shape, concentration, and surface chemistry and influences the properties and performance of the materials. Here a self-assembly strategy is described for fabrication of complex and well-defined BC-NP composites using colloidal gold and silver NPs of different sizes, shapes, and concentrations. The self-assembly process results in nanocomposites with distinct biophysical and optical properties. In addition to antibacterial materials and materials with excellent senor performance, materials with unique mechanoplasmonic properties are developed. The homogenous incorporation of plasmonic gold NPs in the BC enables extensive modulation of the optical properties by mechanical stimuli. Compression gives rise to near-field coupling between adsorbed NPs, resulting in tunable spectral variations and enhanced broadband absorption that amplify both nonlinear optical and thermoplasmonic effects and enables novel biosensing strategies.

The skin is the largest organ of the human body. Wounds disrupt the functions of the skin and can have catastrophic consequences for an individual resulting in significant morbidity and mortality. Wound infections are common and can substantially delay healing and can result in non-healing wounds and sepsis. Early diagnosis and treatment of infection reduce risk of complications and support wound healing. Methods for monitoring of wound pH can facilitate early detection of infection. Here we show a novel strategy for integrating pH sensing capabilities in state-of-the-art hydrogel-based wound dressings fabricated from bacterial nanocellulose (BC). A high surface area material was developed by self-assembly of mesoporous silica nanoparticles (MSNs) in BC. By encapsulating a pH-responsive dye in the MSNs, wound dressings for continuous pH sensing with spatiotemporal resolution were developed. The pH responsive BC-based nanocomposites demonstrated excellent wound dressing properties, with respect to conformability, mechanical properties, and water vapor transmission rate. In addition to facilitating rapid colorimetric assessment of wound pH, this strategy for generating functional BC-MSN nanocomposites can be further be adapted for encapsulation and release of bioactive compounds for treatment of hard-to-heal wounds, enabling development of novel wound care materials.

Green and scalable materials are essential to fulfill the need of electrification for transitioning into a fossil-fuels free society, and sustainability is a requirement for all new technologies. Rechargeable batteries are one of the most important elements for electrification, enabling the transition to mobile electronics, electrical vehicles and grid storage. We here report synthesis and characterization of polyelectrolyte complexes of alginate and chitosan, both biopolymers deriving from the sea, for transport of zinc ions in hydrogel electrolytes. We have used vibrational spectroscopy, thermal measurements and microscopy, as well as transport measurements with ohmic or blocking contacts. The transference number for zinc ions is close to 1, the conductivity is approximate to 10 mS/cm, with stability at Zn interfaces seen through 7000 cycles in symmetric zinc//zinc cell. A zinc ion aqueous electrolyte was prepared from blends of chitosan and alginate, by using a simple and scalable route. These green zinc ion electrolytes exhibit a stability window up to 2 V, a zinc ion transference number close to 1, and electrochemical cyclability over 7000 cycles at interfaces to zinc. This biologically derived polyelectrolyte complex offers many possibilities for optimizing transport and stability at electrode interfaces.image

Materials that provide independent control of infrared thermal radiation and haze in the visible could benefit many areas and applications, including clothing, packaging and photovoltaics. Here, we study this possibility for a metamaterial composite paper based on cellulose nanofibrils (CNF) and silicon dioxide (SiO2) microparticles with infrared (IR) Frohlich phonon resonances. This CNF-SiO2 composite shows outstanding transparency in the visible wavelength range, with the option of controlling light diffusion and haze from almost zero to 90% by varying the SiO2 microparticle concentration. We further show that the transparent metamaterial paper could maintain high thermal emissivity in the atmospheric IR window, as attributed to strong IR absorption of both the nanocellulose and the resonant SiO2 microparticles. The high IR emissivity and low visible absorption make the paper suitable for passive radiative cooling and we demonstrate cooling of the paper to around 3 degrees C below ambient air temperature by exposing it to the sky.

A combined computational and experimental study of small unilamellar vesicle (SUV) fusion on mixed self-assembled monolayers (SAMs) terminated with different deuterated tether moieties (-(CD2)(7)CD3 or -(CD2)(15)CD3) is reported. Tethered bilayer lipid membrane (tBLM) formation of synthetic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine was initially probed on SAMs with controlled tether (d-alkyl tail) surface densities and lateral molecular packing using quartz crystal microbalance with dissipation monitoring (QCM-D). Long time-scale coarse-grained molecular dynamics (MD) simulations were then employed to elucidate the mechanisms behind the interaction between the SUVs and the different phases formed by the -(CD2)(7)CD3 and -(CD2)(15)CD3 tethers. Furthermore, a series of real time kinetics was recorded under different osmotic conditions using QCM-D to determine the accumulated lipid mass and for probing the fusion process. It is shown that the key factors driving the SUV fusion and tBLM formation on this type of surfaces involve tether insertion into the SUVs along with vesicle deformation. It is also evident that surface densities of the tethers as small as a few mol% are sufficient to obtain stable tBLMs with a high reproducibility. The described "sparsely tethered" tBLM system can be advantageous in studying different biophysical phenomena, such as membrane protein insertion, effects of receptor clustering, and raft formation.

Green hydrogen is identified as one of the prime clean energy carriers due to its high energy density and a zero emission of CO2. A possible solution for the transport of H2 in a safe and low-cost way is in the form of liquid organic hydrogen carriers (LOHCs). As an alternative to loading LOHC with H2 via a two-step procedure involving preliminary electrolytic production of H2 and subsequent chemical hydrogenation of the LOHC, we explore here the possibility of electrochemical hydrogen storage (EHS) via conversion of proton of a proton donor into a hydrogen atom involved in covalent bonds with the LOHC (R) via a protoncoupled electron transfer (PCET) reaction: . We chose 9-fluorenone/ fluorenol (Fnone/Fnol) conversion as such a model PCET reaction. The electrochemical activation of Fnone via two sequential electron transfers was monitored with in-situ and operando spectroscopies in absence and in presence of different alcohols as proton donors of different reactivity, which enabled us to both quantify and get the mechanistic insight on PCET. The possibility of hydrogen extraction from the loaded carrier molecule was illustrated by chemical activation.

The emerging stretchable photonics field faces challenges, like the robust integration of optical elements into elastic matrices or the generation of large optomechanical effects. Here, the first stretchable plasmonic-enhanced and wrinkled Fabry-Perot (FP) cavities are demonstrated, which are composed of self-embedded arrays of Au nanostructures at controlled depths into elastomer films. The novel self-embedding process is triggered by the Au nanostructures catalytic activity, which locally increases the polymer curing rate, thereby inducing a mechanical stress that simultaneously pulls the Au nanostructures into the polymer and forms a wrinkled skin layer. This geometry yields unprecedented optomechanical effects produced by the coupling of the broad plasmonic modes of the Au nanostructures and the FP modes, which are modulated by the wrinkled optical cavity. As a result, film stretching induces drastic changes in both the spectral position and intensity of the plasmonic-enhanced FP resonances due to the simultaneous cavity thickness reduction and cavity wrinkle flattening, thus increasing the cavity finesse. These optomechanical effects are exploited to demonstrate new strain-sensing approaches, achieving a strain detection limit of 0.006%, i.e., 16-fold lower than current optical strain-detection schemes.

BACKGROUND Avocado is affected by Colletotrichum gloeosporioides causing anthracnose. Antagonistic microorganisms against C. gloeosporioides represent an alternative for biological control. Accordingly, in the present study, we focused on the isolation and characterization of potential antagonist bacteria against a member of the C. gloeosporioides species complex with respect to their possible future application. RESULTS Samples of avocado rhizospheric soil were aquired from an orchard located in Ocuituco, Morelos, Mexico, aiming to obtain bacterial isolates with potential antifungal activity. From the soil samples, 136 bacteria were isolated and they were then challenged against a member of the C. gloeosporioides species complex; only three bacterial isolates A1, A2 and A3 significantly diminished mycelial fungal growth by 75%, 70% and 60%, respectively. Two of these isolates were identified by 16S rRNA as Bacillus mycoides (A1 and A2) and the third was identified as Bacillus tequilensis (A3). Bacillus mycoides bacterial cell-free supernatant reduced the mycelial growth of a member of the C. gloeosporioides species complex isolated from avocado by 65%, whereas Bacillus tequilensis A3 supernatant did so by 25% after 3 days post inoculation. Bacillus tequilensis mycoides A1 was a producer of proteases, indolacetic acid and siderophores. Preventive treatment using a cell-free supernatant of B. mycoides A1 diminished the severity of anthracnose disease (41.9%) on avocado fruit. CONCLUSION These results reveal the possibility of using B. mycoides A1 as a potential biological control agent. (c) 2020 Society of Chemical Industry

Silk fibers have unique mechanical properties, and many studies of silk aim at understanding how these properties are related to secondary structure content, which often is determined by infrared spectroscopy. We report significant method-induced irreversible structural changes to both natural and synthetic spider silk fibers, derived from the widely used attenuated total reflection Fourier-transform infrared (ATR-FTIR) technique. By varying the force used to bring fibers into contact with the internal reflection elements of ATR-FTIR accessories, we observed correlated and largely irreversible changes in the secondary structure, with shape relaxation under pressure occurring within minutes. Fitting of spectral components shows that these changes agree with transformations from the alpha-helix to the beta-sheet secondary structure with possible contributions from other secondary structure elements. We further confirm the findings with IR microspectroscopy, where similar differences were seen between the pressed and unaffected regions of spider silk fibers. Our findings show that ATR-FTIR spectroscopy requires care in its use and in the interpretation of the results.

Fast and accurate detection of antimicrobial resistance in pathogens remains a challenge, and with the increase in antimicrobial resistance due to mis- and overuse of antibiotics, it has become an urgent public health problem. We demonstrate how infrared attenuated total reflection (IR-ATR) can be used as a simple method for assessment of bacterial susceptibility to antibiotics. This is achieved by monitoring the metabolic activities of bacterial cells via nutrient consumption and using this as an indicator of bacterial viability. Principal component analysis of the obtained spectra provides a tool for fast and simple discrimination of antimicrobial resistance in the acquired data. We demonstrate this concept using four bacterial strains and four different antibiotics, showing that the change in glucose concentration in the growth medium after 2 h, as monitored by IR-ATR, can be used as a spectroscopic diagnostic technique, to reduce detection time and to improve quality in the assessment of antimicrobial resistance in pathogens.

In this research, a fundamental study was conducted on damage behavior of cathodic arc evaporated TiN and Ti0.44Al0.56N coatings, in terms of oxidation and cracking/spallation, when they were exposed to single-pulse laser treatment in a temperature range of 1200-2100 degrees C. Moreover, a multiple-pulse laser treatment was designed to apply thermo-mechanical loads on the coatings in order to evaluate their thermal degradation during rapid heating/cooling cycles between 200 and 1200 degrees C. Single-pulse treatment of TiN up to 1500 degrees C led to the intercolumnar cracking and formation of ultrafine TiO grains. An increase in temperature up to 2100 degrees C resulted in a notable bulging of the surface, and formation of TiO2 of various morphologies such as grainy structure, dense molten and re-solidified structure, droplets from melt expulsion and, more interestingly, nanofibers. Multiplepulse treatment of TiN was accompanied by a severe cracking and spallation, which divided the surface into two layers: a heavily cracked top layer composed of dense TiO2 grains, and a bottom layer having porous TiO2 grains indicating incomplete oxidation. Conversely, Ti0.44Al0.56N did not show any visible cracking and oxidation after single-pulse treatment. Multiple-pulse treatment did not also yield cracking and spallation for Ti0.44Al0.56N, and its ablated region consisted of TiO2 grains combined with thin Al2O3 platelets. An excellent combination of properties including higher oxidation resistance and greater fracture toughness at high temperatures led to a higher thermal damage resistance for Ti0.44Al0.56N coating compared to TiN when undergoing single- and multiple-pulse laser treatments.

Environmentally friendly halide double perovskites with improved stability are regarded as a promising alternative to lead halide perovskites. The benchmark double perovskite, Cs2AgBiBr6, shows attractive optical and electronic features, making it promising for high-efficiency optoelectronic devices. However, the large band gap limits its further applications, especially for photovoltaics. Herein, we develop a novel crystal-engineering strategy to significantly decrease the band gap by approximately 0.26 eV, reaching the smallest reported band gap of 1.72 eV for Cs(2)AgBiBr(6)under ambient conditions. The band-gap narrowing is confirmed by both absorption and photoluminescence measurements. Our first-principles calculations indicate that enhanced Ag-Bi disorder has a large impact on the band structure and decreases the band gap, providing a possible explanation of the observed band-gap narrowing effect. This work provides new insights for achieving lead-free double perovskites with suitable band gaps for optoelectronic applications.

Three-dimensional (3D) cell culture facilitates development of biological relevant assays for drug screening and toxicity testing. Compared to conventional 2D cell culture, cells cultured in 3D can more accurately mimic human tissues and organs and thus provide ex vivo data with potentially better predictive value for cancer research, pharmacology, and toxicology, reducing the need for animal models, improving experimental reproducibility, and reducing time and costs in drug development. The most widely used options for scaffold-based 3D cell culture are, however, based on poorly defined biologically derived extracellular matrix (ECM) with limited possibilities to tailor material properties and that are difficult to combine with state-of-the art biofabrication techniques.   

The overall aim this thesis was to design and explore modular hyaluronan (HA) based ECM-mimicking hydrogels with tuneable physiochemical properties and biofunctionalities, for development of advanced 3D cell models and biofabrication. The thesis work is presented in five papers. In paper I, we used copper free click chemistry for both hydrogel cross-linking and functionalization with fibronectin derived peptide sequences for culture of human induced pluripotent-derived hepatocytes in a perfused microfluidic system. The tuneable and bioorthogonal cross-linking enabled both retention of high cell viabilities and fabrication of a functional liver-on-chip solution. In paper II, we combined the developed HA-based hydrogel system with homo- and heterodimerizing helix-loop-helix peptides for modulation of both cross-linking density and biofunctionalization. We further demonstrated the possibilities to use these hydrogels as bioinks for 3D bioprinting where both the molecular composition and the physical properties of the printed structures could be dynamically altered, providing new avenues for four-dimensional (4D) bioprinting. In paper III we investigated the possibilities to chemically conjugate full size recombinant human laminin-521 (LN521) in the HA-based hydrogels system using copper-free click chemistry, with the aim to enable 3D culture and 3D bioprinting of neurons. We quantified the impact of using different linkers to tether LN521 and the influence of LN-functionalization on the structural and mechanical properties of the hydrogels. We show that both differentiated and non-differentiated neuroblastoma cells and long-term self-renewing neuroepithelial stem cells (lt-NES) remained viable in the hydrogels. The hydrogels also had a protected effect on lt-NES during syringe ejection and bioprinting. In paper IV, we used HA-based hydrogels modified with peptides sequences derived from fibronectin and laminin for culture of fetal primary astrocytes (FPA). We explored both the interactions between the hydrogels and FPA and possibilities to 3D bioprint FPAs.  Finally, in paper V, we developed HA-nanocellulose composite hydrogels with the aim to increase printing fidelity and enable fabrication of multi-layered bioprinted structures without the use of a support bath. In addition to HA, we used wood-fibre derived nanocellulose (NC) to increase the viscosity of the bioink during the printing process.  

The developed biorthogonal and modular hydrogel systems provide a large degree of flexibility that allows for encapsulation and culture of different cell types and processing using different techniques, which can contribute to further exploration of fabrication of biologically relevant tissue and disease models.   

Laminins (LNs) are key components in the extracellular matrix of neuronal tissues in the developing brain and neural stem cell niches. LN-presenting hydrogels can provide a biologically relevant matrix for the 3D culture of neurons toward development of advanced tissue models and cell-based therapies for the treatment of neurological disorders. Biologically derived hydrogels are rich in fragmented LN and are poorly defined concerning composition, which hampers clinical translation. Engineered hydrogels require elaborate and often cytotoxic chemistries for cross-linking and LN conjugation and provide limited possibilities to tailor the properties of the materials. Here a modular hydrogel system for neural 3D cell cultures, based on hyaluronan and poly(ethylene glycol), that is cross-linked and functionalized with human recombinant LN-521 using bioorthogonal copper-free click chemistry, is shown. Encapsulated human neuroblastoma cells demonstrate high viability and grow into spheroids. Long-term neuroepithelial stem cells (lt-NES) cultured in the hydrogels can undergo spontaneous differentiation to neural fate and demonstrate significantly higher viability than cells cultured without LN. The hydrogels further support the structural integrity of 3D bioprinted structures and maintain high viability of bioprinted and syringe extruded lt-NES, which can facilitate biofabrication and development of cell-based therapies.

The current standard method of diagnosing coronavirus disease 2019 (COVID-19) involves uncomfortable and invasive nasopharyngeal (NP) sampling using cotton swabs (CS), which can be unsuitable for self-testing. Although mid-turbinate sampling is an alternative, it has a lower diagnostic yield than NP sampling. Nasal wash (NW) has a similar diagnostic yield to NP sampling, but is cumbersome to perform. In this study, we introduce a 3D printed fluidic swab (3DPFS) that enables easy NW sampling for COVID-19 testing with improved diagnostic yield. The 3DPFS comprises a swab head, microchannel, and socket that can be connected to a syringe containing 250 & mu;L of NW solution. The 3DPFS efficiently collects nasal fluid from the surface of the nasal cavity, resulting in higher sensitivity than CS for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This was confirmed by both reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and lateral flow assays (LFA) in virus-spiked nasal samples and clinical samples. Additionally, users reported greater comfort when using the 3DPFS compared to CS. These findings suggest that the 3DPFS can improve the performance of COVID-19 testing by facilitating efficient and less painful nasal sample collection.

Extracellular vesicles (EVs) are a group of membrane vesicles that play important roles in cell-to-cell and interspecies/interkingdom communications by modulating the pathophysiological conditions of recipient cells. Recent evidence has implied their potential roles in the gut-brain axis (GBA), which is a complex bidirectional communication system between the gut environment and brain pathophysiology. Despite the evidence, the roles of EVs in the gut microenvironment in the GBA are less highlighted. Moreover, there are critical challenges in the current GBA models and analyzing techniques for EVs, which may hinder the research. Currently, advances in organ-on-a-chip (OOC) technologies have provided a promising solution. Here, we review the potential effects of EVs occurring in the gut environment on brain physiology and behavior and discuss how to apply OOCs to research the GBA mediated by EVs in the gut microenvironment.

The rapid growth of wearables has created a demand for lightweight, elastic and conformal energy harvesting and storage devices. The conducting polymer poly(3,4-ethylenedioxythiophene) has shown great promise for thermoelectric generators, however, the thick layers of pristine poly(3,4-ethylenedioxythiophene) required for effective energy harvesting are too hard and brittle for seamless integration into wearables. Poly(3,4-ethylenedioxythiophene)-elastomer composites have been developed to improve its mechanical properties, although so far without simultaneously achieving softness, high electrical conductivity, and stretchability. Here we report an aqueously processed poly(3,4-ethylenedioxythiophene)-polyurethane-ionic liquid composite, which combines high conductivity (>140Scm(-1)) with superior stretchability (>600%), elasticity, and low Youngs modulus (<7MPa). The outstanding performance of this organic nanocomposite is the result of favorable percolation networks on the nano- and micro-scale and the plasticizing effect of the ionic liquid. The elastic thermoelectric material is implemented in the first reported intrinsically stretchable organic thermoelectric module. Though deformable thermoelectric materials are desirable for integrating thermoelectric devices into wearable electronics, typical thermoelectric materials are too brittle for practical application. Here, the authors report a high-performance elastic composite for stretchable thermoelectric modules.

Sea stars can adhere to various underwater substrata using an adhesive secretion of which Sfp1 is a major component. Sfp1 is a multimodular protein composed of four subunits (Sfp1 Alpha, Beta, Delta, and Gamma) displaying different functional domains. We recombinantly produced two fragments of Sfp1 comprising most of its functional domains: the C-terminal part of the Beta subunit (rSfp1 Beta C-term) and the Delta subunit (rSfp1 Delta). Surface plasmon resonance analyses of protein adsorption onto different model surfaces showed that rSfp1 Beta C-term exhibits a significantly higher adsorption than the fibrinogen control on hydrophobic, hydrophilic protein-resistant, and charged self-assembled monolayers, while rSfp1 Delta adsorbed more on negatively charged and on protein-resistant surfaces compared to fibrinogen. Truncated recombinant rSfp1 Beta C-term proteins were produced in order to investigate the role of the different functional domains in the adsorption of this protein. The analysis of their adsorption capacities on glass showed that two mechanisms are involved in rSfp1 Beta C-term adsorption: (1) one mediated by the EGF-like domain and involving Ca2+ and Mg2+ ions, and (2) one mediated by the sequence of Sfp1 Beta with no homology with known functional domain in databases, in the presence of Na+, Ca2+ and Mg2+ ions.

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This minireview suggests a conceptual and user-oriented approach for the design of process monitoring systems in bioprocessing. Advancement of process analytical techniques for quantification of critical analytes can take advantage of basic conceptual process design to support reasoning, reconsidering and ranking solutions. Issues on analysis in complex bio-industrial media, sensitivity and selectivity are highlighted from users perspectives. Meeting challenging analytical demands for understanding the critical interplay between the emerging bioprocesses, their biomolecular complexity and the needs for user-friendly analytical tools are discussed. By that, a thorough design approach is suggested based on a holistic design thinking in the quest for better analytical opportunities to solve established and emerging analytical needs.

Astrocytes play an important role in the central nervous system, contributing to the development of and maintenance of synapses, recycling of neurotransmitters, and the integrity and function of the blood-brain barrier. Astrocytes are also linked to the pathophysiology of various neurodegenerative diseases. Astrocyte function and organization are tightly regulated by interactions mediated by the extracellular matrix (ECM). Engineered hydrogels can mimic key aspects of the ECM and can allow for systematic studies of ECM-related factors that govern astrocyte behaviour. In this study, we explore the interactions between neuroblastoma (SH-SY5Y) and glioblastoma (U87) cell lines and human fetal primary astrocytes (FPA) with a modular hyaluronan-based hydrogel system. Morphological analysis reveals that FPA have a higher degree of interactions with the hyaluronan-based gels compared to the cell lines. This interaction is enhanced by conjugation of cell-adhesion peptides (cRGD and IKVAV) to the hyaluronan backbone. These effects are retained and pronounced in 3D bioprinted structures. Bioprinted FPA using cRGD functionalized hyaluronan show extensive and defined protrusions and multiple connections between neighboring cells. Possibilities to tailor and optimize astrocyte-compatible ECM-mimicking hydrogels that can be processed by means of additive biofabrication can facilitate the development of advanced tissue and disease models of the central nervous system.

Biogas production from manure is attractive to support plans towards a circular economy as it allows for renewable energy production and nutrient recycling in agriculture. Finding optimal locations for biogas plants, which minimize transport distances to and from farms, while accounting for multiple feasibility constraints, remains a challenge. We developed 1 km2 spatially-explicit datasets for known feasibility constraints such as distance to housing, compatible land-use zoning, and the presence of roads with sufficient weight bearing capacity. These datasets were used to improve the realism of an optimization model designed to minimize transportation costs in Sweden. At a national level, the presence of durable enough roads most limited the number of suitable locations for a plant. We further focused our analysis on a case study region where a company wanted to invest in a new manure-based biogas plant. In contrast to the national level, the constraint for remaining at least 500 m from housing/buildings had the greatest limiting impact, excluding 71% of grids in the Sjo center dot bo or Tomelilla municipalities of Southern Sweden. Still, we identified 105 suitable locations for a new biogas plant. The most suitable location, accounting for feasibility and minimized transportation costs, changed when simultaneously accounting for another proposed plant in a neighboring municipality. Our results indicate that utilizing both local and national datasets can help narrow down potential biogas development sites and that long-term planning is necessary for actors with ambitions to build several plants to minimize costs and maximize energy and nutrient recycling benefits.

Multidrug resistance bacteria constitue an increasing global health problem and the development of novel therapeutic strategies to face this challenge is urgent. Antimicrobial peptides have been proven as potent agents against pathogenic bacteria shown by promising in vitro results. The aim of this study was to characterize the antimicrobial effects of PLNC8 alpha beta on cell signaling pathways and inflammatory responses of human keratinocytes infected with S. aureus. PLNC8 alpha beta did not affect the viability of human keratinocytes but upregulated several cytokines (IL-1 beta, IL-6, CXCL8), MMPs (MMP1, MMP2, MMP9, MMP10) and growth factors (VEGF and PDGF-AA), which are essential in cell regeneration. S. aureus induced the expression of several inflammatory mediators at the gene and protein level and PLNC8 alpha beta was able to significantly suppress these effects. Intracellular signaling events involved primarily c-Jun via JNK, c-Fos and NF kappa B, suggesting their essential role in the initiation of inflammatory responses in human keratinocytes. PLNC8 alpha beta was shown to modulate early keratinocyte responses, without affecting their viability. The peptides have high selectivity towards S. aureus and were efficient at eliminating the bacteria and counteracting their inflammatory and cytotoxic effects, alone and in combination with low concentrations of gentamicin. We propose that PLNC8 alpha beta may be developed to combat infections caused by Staphylococcus spp.

Engineered human tissue and disease models can decrease the cost and time of developing new drugs and treatments, facilitate personalized medicine, and eliminate the need for animal models that poorly represent the human body and are ethically problematic. However, the current conventional cell expansion methods using 2D culture flasks cannot enable the development of such complex multi-cellular 3D models. In general, hydrogels are considered promising materials that can make the biofabrication of tissue models possible. Hydrogels are highly hydrated materials comprised of either synthetic or naturally derived polymers, or a combination of both, and can form an environment mimicking the biomacromolecular network surrounding cells in the body. This network of biopolymers, known as extracellular matrix (ECM), is comprised of proteins such as collagen, laminin, fibronectin, and polysaccharides such as hyaluronan (HA), heparan, keratan, and chondroitin sulfate. The design of hydrogels representing the physical and biochemical properties of the ECM and which can be used for biofabrication is challenging but of increasing interest due to the rapid progress in the development of 3D and 4D bioprinting techniques. As the ECM properties differ between various tissues and disease conditions and change over time, a dynamic modular hydrogel system is needed to that can be optimized for each cell and tissue type. This thesis aims to develop modular enzyme-responsive polysaccharide-based hydrogels for 3D cell culture and biofabrication. The natural polysaccharides, hyaluronic acid (HA) and alginate (Alg) were used as the main backbone in the hydrogels developed in this thesis. HA was modified by conjugating bicyclo[6.1.0]non-4-yne (BCN) to the backbone to form HA-BCN-based hydrogels by a bioorthogonal strain-promoted alkyne-azide cycloaddition. The click reaction between BCN and azide groups allowed for modulating the biochemical and mechanical properties of the HA-BCN hydrogels. HA-BCN was further decorated with peptides to explore peptide folding and dimerization-mediated dynamic cross-linking and biofunctionalization. This system was further used to explore possibilities to dynamically alter the properties of 3D bioprinted structures, mimicking the biomineralization process in bone tissue. In a different study, a tumor model comprising fibroblast and breast cancer cells (MCF7) was bioprinted using HA-BCN cross-linked by matrix metalloporotease (MMP) cleavable and PEG-diazide MMP-resistant cross-linkers, demonstrating the synergistic relationship between hydrogel degradability and cancer cell growth, intensified by the presence of fibroblasts. The possibility of incorporating a conductive module into this hydrogel system was explored using the enzyme-assisted polymerization of ETE-S to form an interpenetrating conductive network inside HA-BCN hydrogel. The in situ and user-triggered polymerization of conductive ETE-S was demonstrated after 3D printing HA-BCN bioink containing ETE-S monomers into a lattice shape structure. We also demonstrated that cellulose nanofibrils (CNF) improved the printability of HA-BCN bioinks, and this hybrid bioink was used for printing self-standing cell-laden 3D structures. Besides these studies, a novel enzymatically triggered thiol-based chemistry was developed to address the unwanted oxidation of thiol-containing hydrogels and decrease the off-target thiol reactions during hydrogel synthesis and formation. Alginate containing sulfhydryl moieties, protected by an enzyme-labile Phacm group (AlgCP), was treated with penicillin G acylase and subsequently formed a disulfide cross-linked hydrogel. We studied the gelation kinetics and rheological properties of AlgCP and different modes of cross-linking by reversible disulfide bonds, a thiol-maleimide Micheale-type addition reaction, and ionic interactions between alginate and Ca²⁺ ions. MCF7 breast cancer cells cultured in the AlgCP hydrogels formed spheroids that could be harvested by GSH dissolution of the hydrogels. Finally, this novel chemistry enabled bioprinting of multi-material 3D structures with control over the printed structure's physiochemical properties, including the type and density of cross-linkers. Bioprinted fibroblasts formed extended morphology, and MCF7 cells formed spheroids in the bioprinted lattice structures.   

The hydrogel systems developed and explored in this thesis are modular and exhibit dynamic and tunable properties, and are applicable for a wide range of 3D cell culture and bioprinting applications. The hydrogels were either formed in response to the activity of an enzyme or remodeled by enzymes. Both enzyme-responsive HA-BCN and AlgCP hydrogel systems are promising bioinks for generating more elaborate and spatially defined cell-laden 3D structures whose features can be altered post-printing by cell-secreted and extrinsic reagents. These hydrogel-based toolkits can play a vital role in developing tissue and disease models that can make the drug discovery process faster, cheaper, and animal-free. 

Thiolated polymers are widely used in hydrogels for drug delivery, tissue engineering, and biofabrication. The oxidation of thiols is spontaneous, resulting in the formation of disulfide bridges and cross linking of polymers. The cross-linking process is, however, difficult to control and is initiated directly when the thiolated components are exposed to ambient conditions, which significantly complicates handling of the materials. Here, we show a fully bioorthogonal enzyme-mediated thiol-based chemistry for dynamic covalent cross-linking of carbohydrate-based hydrogels that circumvents the problems with uncontrolled thiol oxidation. Alginate was modified with cysteine residues, protected by an enzyme-labile thiol-protecting group (Phacm). Releasing the Phacm group by penicillin G acylase generates free thiols that oxidize under physiological conditions, resulting in a reversible cross-linking and formation of hydrogels with tunable stiffness. Prior to deprotection, the components can be exposed to ambient conditions. The enzyme-triggered deprotection and subsequent gelation allows for encapsulation of cells and 3D bioprinting of cell-laden hydrogel structures. Remaining deprotected thiols enabled postprinting modifications and hydrogel self-healing. The proposed hydrogel synthesis strategy significantly increases the versatility of thiol-based cross-linking chemistries and provides new possibilities to generate dynamic covalent hydrogels for a broad range of biomedical applications.

Polymer coatings are inexpensive surface modifications providing a wide variety of functions. There is an ever-present motivation to improve the films’ performance and to decrease the cost and the environmental footprint of their production. This thesis includes the study of the structural and functional parameters of polymer coatings that are aimed at preventing biological fouling, the unwanted attachment of organic molecules and organisms on surfaces. The focus was on thin films prepared by the self-initiated photografting and photopolymerization method. This is a UV-initiated polymerization reaction that does not require additional chemicals beyond the monomers and the solvent. Since biofouling is a prominent problem in wet environments, the emphasis was placed on the hydrated structure of the films. Neutron reflectometry was selected as a primary method for these studies, since it is a powerful method for investigating the structure of polymer thin films, especially in the hydrated state due to the labelling offered by isotope substitution. This allows the determination of the solvent volume fraction depth profile, which reveals the chain segment density profile in the hydrated film. To resolve fast changes in the film structure and to study the chemical composition, spectroscopic ellipsometry and infrared absorption spectroscopy was implemented in a setup for in-situ measurements in parallel with neutron reflectometry.  

This thesis contains an introduction and five research articles, and it can be divided into two main parts: the first focusing on the polymerization reaction and the resulting polymer films and the second on the reflectivity method and instrumentation development. Firstly, uncharged hydrophilic polymer layers were prepared by self-initiated photografting and photopolymerization and the hydrated structure of these was investigated. It was found that the films follow a stretched profile indicating negligible crosslinking, and that the growth dynamics is determined by the balance of grafting and removal through radiation damage. Studying sequential grafting of oppositely charged polyelectrolytes confirmed the results on growth dynamics and showed the effects of electrostatic interactions between the monomers. This also demonstrated that the polymerization method is ill suited for preparing block co-polymers due to the removal of material from the previous layer. However, these studies also show that the growth of the second layer tends to proceed from the substrate, forming a system where the two kinds of chains co-exist and interact in the same layer. The grafting of random co-polymers was also investigated by comparing the anti-fouling performance of layers made from a mixture of oppositely charged monomers to layers made using zwitterionic polymers, resulting in no significant difference. This was attributed to the pairwise deposition of oppositely charged monomers, further emphasizing the importance of the Coulomb force in defining the structure of the charged films. The second part of this work focuses on instrument development. Here the building and testing of an angle-dispersive reflectometer is presented, and the design and first applications of an in-situ setup for measuring spectroscopic ellipsometry and infrared spectroscopy along with neutron reflectometry is described. 

By investigating the structure of the polymer films prepared by self-initiated photografting and photopolymerization, this work improved the understanding of this method, facilitating the development of new applications in the future. By combining additional methods with neutron reflectometry, both fast changes in the structure and the chemical evolution of the samples can be investigated. However, the differences in the sensitivities and the structural models required by the probes present new challenges in modelling. 

We have prepared a series of ampholytic polymer films, using a self-initiated photografting and photopolymerization (SI-PGP) method to sequentially polymerize first anionic (deuterated methacrylic acid (dMAA)) and thereafter cationic (2-aminoethyl methacrylate (AEMA)) monomers to investigate the SI-PGP grafting process. Dry films were investigated by ellipsometry, X-ray, and neutron reflectometry, and their swelling was followed over a pH range from 4.5 to 10.5 with spectroscopic ellipsometry. The deuterated monomer allows us to separate the distributions of the two components by neutron reflectometry. Growth of both polymers proceeds via grafting of solution-polymerized fragments to the surface, and also the second layer is primarily grafted to the substrate and not as a continuation of the existing chains. The polymer films are stratified, with one layer of near 1:1 composition and the other layer enriched in one component and located either above or below the former layer. The ellipsometry results show swelling transitions at low and high pH but with no systematic variation in the pH values where these transitions occur. The results suggest that grafting density in SI-PGP-prepared homopolymers could be increased via repeated polymerization steps, but that this process does not necessarily increase the average chain length.