Minimally designed thermo-magnetic dual responsive soft robots for complex applications.

The fabrication of thermo-magnetic dual-responsive soft robots often requires intricate designs to implement complex locomotion patterns and utilize the implemented responsive behaviors. This work demonstrates a minimally designed soft robot based on poly-N-isopropylacrylamide (pNIPAM) and ferromagnetic particles, showcasing excellent control over both thermo- and magnetic responses. Free radical polymerization enables the magnetic particles to be entrapped homogeneously within the polymeric network. The integration of magnetic shape programming and temperature response allows the robot to perform various tasks including shaping, locomotion, pick-and-place, and release maneuvers of objects using independent triggers. The robot can be immobilized in a gripping state through magnetic actuation, and a subsequent increase in temperature transitions the robot from a swollen to a collapsed state. The temperature switch enables the robot to maintain a secured configuration while executing other movements via magnetic actuation. This approach offers a straightforward yet effective solution for achieving full control over both stimuli in dual-responsive soft robotics.

Layered Double Hydroxides: Recent Progress and Promising Perspectives Toward Biomedical Applications.

Layered double hydroxides (LDHs) have been widely studied for biomedical applications due to their excellent properties, such as good biocompatibility, degradability, interlayer ion exchangeability, high loading capacity, pH-responsive release, and large specific surface area. Furthermore, the flexibility in the structural composition and ease of surface modification of LDHs makes it possible to develop specifically functionalized LDHs to meet the needs of different applications. In this review, the recent advances of LDHs for biomedical applications, which include LDH-based drug delivery systems, LDHs for cancer diagnosis and therapy, tissue engineering, coatings, functional membranes, and biosensors, are comprehensively discussed. From these various biomedical research fields, it can be seen that there is great potential and possibility for the use of LDHs in biomedical applications. However, at the same time, it must be recognized that the actual clinical translation of LDHs is still very limited. Therefore, the current limitations of related research on LDHs are discussed by combining limited examples of actual clinical translation with requirements for clinical translation of biomaterials. Finally, an outlook on future research related to LDHs is provided.

Sono-processes: Emerging systems and their applicability within the (bio-)medical field.

Sonochemistry, although established in various fields, is still an emerging field finding new effects of ultrasound on chemical systems and are of particular interest for the biomedical field. This interdisciplinary area of research explores the use of acoustic waves with frequencies ranging from 20 kHz to 1 MHz to induce physical and chemical changes. By subjecting liquids to ultrasonic waves, sonochemistry has demonstrated the ability to accelerate reaction rates, alter chemical reaction pathways, and change physical properties of the system while operating under mild reaction conditions. It has found its way into diverse industries including food processing, pharmaceuticals, material science, and environmental remediation. This review provides an overview of the principles, advancements, and applications of sonochemistry with a particular focus on the domain of (bio-)medicine. Despite the numerous benefits sonochemistry has to offer, most of the research in the (bio-)medical field remains in the laboratory stage. Translation of these systems into clinical practice is complex as parameters used for medical ultrasound are limited and toxic side effects must be minimized in order to meet regulatory approval. However, directing attention towards the applicability of the system in clinical practice from the early stages of research holds significant potential to further amplify the role of sonochemistry in clinical applications.

Collagen type I alters the proteomic signature of macrophages in a collagen morphology-dependent manner.

Idiopathic pulmonary fibrosis is a progressive lung disease that causes scarring and loss of lung function. Macrophages play a key role in fibrosis, but their responses to altered morphological and mechanical properties of the extracellular matrix in fibrosis is relatively unexplored. Our previous work showed functional changes in murine fetal liver-derived alveolar macrophages on fibrous or globular collagen morphologies. In this study, we applied differential proteomics to further investigate molecular mechanisms underlying the observed functional changes. Macrophages cultured on uncoated, fibrous, or globular collagen-coated plastic were analyzed by liquid chromatography-mass spectrometry. The presence of collagen affected expression of 77 proteins, while 142 were differentially expressed between macrophages grown on fibrous or globular collagen. Biological process and pathway enrichment analysis revealed that culturing on any type of collagen induced higher expression of enzymes involved in glycolysis. However, this did not lead to a higher rate of glycolysis, probably because of a concomitant decrease in activity of these enzymes. Our data suggest that macrophages sense collagen morphologies and can respond with changes in expression and activity of metabolism-related proteins. These findings suggest intimate interactions between macrophages and their surroundings that may be important in repair or fibrosis of lung tissue.

Nanogel-based coating as an alternative strategy for biofilm control in drinking water distribution systems.

Biofilm formation and detachment in drinking water distribution systems (DWDS) can lead to several operational issues. Here, an alternative biofilm control strategy of limiting bacterial adhesion by application of a poly(N-isopropylmethacrylamide)-based nanogel coating on DWDS pipe walls was investigated. The nanogel coatings were successfully deposited on surfaces of four polymeric pipe materials commonly applied in DWDS construction. Nanogel-coated and non-coated pipe materials were characterized in terms of their surface hydrophilicity and roughness. Four DWDS relevant bacterial strains, representing Sphingomonas and Pseudomonas, were used to evaluate the anti-adhesive performance of the coating in 4 h adhesion and 24 h biofilm assays. The presence of the nanogel coating resulted in adhesion reduction up to 97%, and biofilm reduction up to 98%, compared to non-coated surfaces. These promising results motivate further investigation of nanogel coatings as a strategy for biofilm prevention in DWDS.

Zwitterionic poly(sulfobetaine methacrylate)-based hydrogel coating for drinking water distribution systems to inhibit adhesion of waterborne bacteria.

Presence of biofilms in drinking water distribution systems (DWDS) can be a nuisance, leading to several operational and maintenance issues (i.e., increased secondary disinfectants demand, pipe damage or increased flow resistance), and so far, no single control practice was found to be sufficiently effective. Here, we propose poly (sulfobetaine methacrylate) (P(SBMA))-based hydrogel coating application as a biofilm control strategy in DWDS. The P(SBMA) coating was synthetized through photoinitiated free radical polymerization on polydimethylsiloxane with different combinations of SBMA as a monomer, and N, N'-methylenebis (acrylamide) (BIS) as a cross-linker. The most stable coating in terms of its mechanical properties was obtained using 20% SBMA with a 20:1 SBMA:BIS ratio. The coating was characterized using Scanning Electron Microscopy, Energy Dispersive X-Ray Spectroscopy, and water contact angle measurements. The anti-adhesive performance of the coating was evaluated in a parallel-plate flow chamber system against adhesion of four bacterial strains representing genera commonly identified in DWDS biofilm communities, Sphingomonas and Pseudomonas. The selected strains exhibited varying adhesion behaviors in terms of attachment density and bacteria distribution on the surface. Despite these differences, after 4 h, presence of the P(SBMA)-based hydrogel coating significantly reduced the number of adhering bacteria by 97%, 94%, 98% and 99%, for Sphingomonas Sph5, Sphingomonas Sph10, Pseudomonas extremorientalis and Pseudomonas aeruginosa, respectively, compared to non-coated surfaces. These findings motivate further research into a potential application of a hydrogel anti-adhesive coating as a localized biofilm control strategy in DWDS, especially on materials known to promote excessive biofilm growth.

Bioinspired Processing: Complex Coacervates as Versatile Inks for 3D Bioprinting.

3D bioprinting is a powerful fabrication technique in biomedical engineering, which is currently limited by the number of available materials that meet all physicochemical and cytocompatibility requirements for biomaterial inks. Inspired by the key role of coacervation in the extrusion and spinning of many natural materials, hyaluronic acid-chitosan complex coacervates are proposed here as tunable biomaterial inks. Complex coacervates are obtained through an associative liquid-liquid phase separation driven by electrostatic attraction between oppositely charged macromolecules. They offer bioactive properties and facile modulation of their mechanical properties through mild physicochemical changes in the environment, making them attractive for 3D bioprinting. Fine-tuning the salt concentration, pH, and molecular weight of the constituent polymers results in biomaterial inks that are printable in air and water. The biomaterial ink, initially a viscoelastic fluid, transitions into a viscoelastic solid upon printing due to dehydration (for printing in air) or due to a change in pH and ionic composition (for printing in solution). Consequently, scaffolds printed using the complex coacervate inks are stable without the need for post-printing processing. Fabricated cell culture scaffolds are cytocompatible and show long-term topological stability. These results pave the way to a new class of easy-to-handle tunable biomaterials for biofabrication.

Tailorable and Biocompatible Supramolecular-Based Hydrogels Featuring two Dynamic Covalent Chemistries.

Dynamic covalent chemistry (DCC) has proven to be a valuable tool in creating fascinating molecules, structures, and emergent properties in fully synthetic systems. Here we report a system that uses two dynamic covalent bonds in tandem, namely disulfides and hydrazones, for the formation of hydrogels containing biologically relevant ligands. The reversibility of disulfide bonds allows fiber formation upon oxidation of dithiol-peptide building block, while the reaction between NH-NH2 functionalized C-terminus and aldehyde cross-linkers results in a gel. The same bond-forming reaction was exploited for the "decoration" of the supramolecular assemblies by cell-adhesion-promoting sequences (RGD and LDV). Fast triggered gelation, cytocompatibility and ability to "on-demand" chemically customize fibrillar scaffold offer potential for applying these systems as a bioactive platform for cell culture and tissue engineering.

Alginate based hydrogel inks for 3D bioprinting of engineered orthopedic tissues.

3D printed hydrogels have emerged as a novel tissue engineering and regeneration platform due to their ability to provide a suitable environment for cell growth. To obtain a well-defined scaffold with good post-printing shape fidelity, a proper hydrogel ink formulation plays a crucial role. In this regard, alginate has received booming interest owing to its biocompatibility, biodegradability, easy functionalization, and fast gelling behavior. Hence, this review highlights the significance of alginate-based hydrogel inks for fabricating 3D printed scaffolds for bone and cartilage regeneration. Herein, we discuss the fundamentals of direct extrusion 3D bioprinting method and provide a comprehensive overview of various alginate-based hydrogel ink formulations that have been used so far. We also summarize the requirements of hydrogel inks and 3D printed scaffolds to achieve similarity to the native tissue environment. Finally, we discuss the challenges, and research directions relevant for future clinical translation.

The Unfolded Protein Response Sensor PERK Mediates Stiffness-Dependent Adaptation in Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor in adults. In addition to genetic causes, the tumor microenvironment (TME), including stiffening of the extracellular matrix (ECM), is a main driver of GBM progression. Mechano-transduction and the unfolded protein response (UPR) are essential for tumor-cell adaptation to harsh TME conditions. Here, we studied the effect of a variable stiff ECM on the morphology and malignant properties of GBM stem cells (GSCs) and, moreover, examined the possible involvement of the UPR sensor PERK herein. For this, stiffness-tunable human blood plasma (HBP)/alginate hydrogels were generated to mimic ECM stiffening. GSCs showed stiffness-dependent adaptation characterized by elongated morphology, increased proliferation, and motility which was accompanied by F-Actin cytoskeletal remodeling. Interestingly, in PERK-deficient GSCs, stiffness adaptation was severely impaired, which was evidenced by low F-Actin levels, the absence of F-Actin remodeling, and decreased cell proliferation and migration. This impairment could be linked with Filamin-A (FLN-A) expression, a known interactor of PERK, which was strongly reduced in PERK-deficient GSCs. In conclusion, we identified a novel PERK/FLNA/F-Actin mechano-adaptive mechanism and found a new function for PERK in the cellular adaptation to ECM stiffening.

Full humanization of the glycolytic pathway in Saccharomyces cerevisiae.

Although transplantation of single genes in yeast plays a key role in elucidating gene functionality in metazoans, technical challenges hamper humanization of full pathways and processes. Empowered by advances in synthetic biology, this study demonstrates the feasibility and implementation of full humanization of glycolysis in yeast. Single gene and full pathway transplantation revealed the remarkable conservation of glycolytic and moonlighting functions and, combined with evolutionary strategies, brought to light context-dependent responses. Human hexokinase 1 and 2, but not 4, required mutations in their catalytic or allosteric sites for functionality in yeast, whereas hexokinase 3 was unable to complement its yeast ortholog. Comparison with human tissues cultures showed preservation of turnover numbers of human glycolytic enzymes in yeast and human cell cultures. This demonstration of transplantation of an entire essential pathway paves the way for establishment of species-, tissue-, and disease-specific metazoan models.

Physics of Brain Cancer: Multiscale Alterations of Glioblastoma Cells under Extracellular Matrix Stiffening.

The biology and physics underlying glioblastoma is not yet completely understood, resulting in the limited efficacy of current clinical therapy. Recent studies have indicated the importance of mechanical stress on the development and malignancy of cancer. Various types of mechanical stress activate adaptive tumor cell responses that include alterations in the extracellular matrix (ECM) which have an impact on tumor malignancy. In this review, we describe and discuss the current knowledge of the effects of ECM alterations and mechanical stress on GBM aggressiveness. Gradual changes in the brain ECM have been connected to the biological and physical alterations of GBM cells. For example, increased expression of several ECM components such as glycosaminoglycans (GAGs), hyaluronic acid (HA), proteoglycans and fibrous proteins result in stiffening of the brain ECM, which alters inter- and intracellular signaling activity. Several mechanosensing signaling pathways have been identified that orchestrate adaptive responses, such as Hippo/YAP, CD44, and actin skeleton signaling, which remodel the cytoskeleton and affect cellular properties such as cell-cell/ECM interactions, growth, and migration/invasion of GBM cells. In vitro, hydrogels are used as a model to mimic the stiffening of the brain ECM and reconstruct its mechanics, which we also discuss. Overall, we provide an overview of the tumor microenvironmental landscape of GBM with a focus on ECM stiffening and its associated adaptive cellular signaling pathways and their possible therapeutic exploitation.

Topography-Mediated Enhancement of Nonviral Gene Delivery in Stem Cells.

Gene delivery holds great promise for bioengineering, biomedical applications, biosensors, diagnoses, and gene therapy. In particular, the influence of topography on gene delivery is considered to be an attractive approach due to low toxicity and localized delivery properties. Even though many gene vectors and transfection systems have been developed to enhance transfection potential and combining it with other forms of stimulations could even further enhance it. Topography is an interesting surface property that has been shown to stimulate differentiation, migration, cell morphology, and cell mechanics. Therefore, it is envisioned that topography might also be able to stimulate transfection. In this study, we tested the hypothesis "topography is able to regulate transfection efficiency", for which we used nano- and microwave-like topographical substrates with wavelengths ranging from 500 nm to 25 µm and assessed the transfectability of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and myoblasts. For transfection, Lipofectamine 2000 and a gene encoding plasmid for red-fluorescent protein (m-Cherry) were used and topography-induced cell morphology and transfection efficiency was analyzed. As a result, topography directs cell spreading, elongation, and proliferation as well as the transfection efficiency, which were investigated but were found not to be correlated and dependent on the cell type. A 55% percent improvement of transfection efficiency was identified for hBM-MSCs grown on 2 µm wrinkles (24.3%) as compared to hBM-MSCs cultured on flat controls (15.7%). For myoblast cells, the highest gene-expression efficiency (46.1%) was observed on the 10 µm topography, which enhanced the transfection efficiency by 64% as compared to the flat control (28.1%). From a qualitative assessment, it was observed that the uptake capacity of cationic complexes of TAMRA-labeled oligodeoxynucleotides (ODNs) was not topography-dependent but that the intracellular release was faster, as indicated by the positively stained nuclei on 2 µm for hBM-MSCs and 10 µm for myoblasts. The presented results indicate that topography enhances the gene-delivery capacity and that the responses are dependent on cell type. This study demonstrates the important role of topography on cell stimulation for gene delivery as well as understanding the uptake capacity of lipoplexes and may be useful for developing advanced nonviral gene delivery strategies.

Dynamic Covalent Cross-Linked Nanogel-Stabilized Pickering Emulsion for Responsive Microstructures.

Designing new dynamic matrices in combination with a highly diverse material formation approach as Pickering emulsions provides the tools to engineer innovative dynamic porous microstructures in a highly controllable fashion. Here, nanogels (nGels) are used, which exhibit dynamic covalent cross-linking capabilities, as surface stabilizing agents in view of their highly controllable physiochemical properties. The method provides the successful formation of dynamic covalent cross-linked hydrogel microstructures based on ketone and amine-functionalized nGels using Pickering emulsions. In this system, a pH-triggerable responsive behavior is incorporated. The physiochemical properties of the resulting microstructure can be further tailored by modifying the intramolecular interactions at the interface, making these systems interesting for a wide range of applications.

Photoactuating Artificial Muscles of Motor Amphiphiles as an Extracellular Matrix Mimetic Scaffold for Mesenchymal Stem Cells.

Mimicking the native extracellular matrix (ECM) as a cell culture scaffold has long attracted scientists from the perspective of supramolecular chemistry for potential application in regenerative medicine. However, the development of the next-generation synthetic materials that mimic key aspects of ECM, with hierarchically oriented supramolecular structures, which are simultaneously highly dynamic and responsive to external stimuli, remains a major challenge. Herein, we present supramolecular assemblies formed by motor amphiphiles (MAs), which mimic the structural features of the hydrogel nature of the ECM and additionally show intrinsic dynamic behavior that allow amplifying molecular motions to macroscopic muscle-like actuating functions induced by light. The supramolecular assembly (named artificial muscle) provides an attractive approach for developing responsive ECM mimetic scaffolds for human bone marrow-derived mesenchymal stem cells (hBM-MSCs). Detailed investigations on the photoisomerization by nuclear magnetic resonance and UV-vis absorption spectroscopy, assembled structures by electron microscopy, the photoactuation process, structural order by X-ray diffraction, and cytotoxicity are presented. Artificial muscles of MAs provide fast photoactuation in water based on the hierarchically anisotropic supramolecular structures and show no cytotoxicity. Particularly important, artificial muscles of MAs with adhered hBM-MSCs still can be actuated by external light stimulation, showing their ability to convert light energy into mechanical signals in biocompatible systems. As a proof-of-concept demonstration, these results provide the potential for building photoactuating ECM mimetic scaffolds by artificial muscle-like supramolecular assemblies based on MAs and offer opportunities for signal transduction in future biohybrid systems of cells and MAs.

Single Cell Reactomics: Real-Time Single-Cell Activation Kinetics of Optically Trapped Macrophages.

Macrophages are well known for their role in immune responses and tissue homeostasis. They can polarize towards various phenotypes in response to biophysical and biochemical stimuli. However, little is known about the early kinetics of macrophage polarization in response to single biophysical or biochemical stimuli. Our approach, combining optical tweezers, confocal fluorescence microscopy, and microfluidics, allows us to isolate single macrophages and follow their immediate responses to a biochemical stimulus in real-time. This strategy enables live-cell imaging at high spatiotemporal resolution and omits surface adhesion and cell-cell contact as biophysical stimuli. The approach is validated by successfully following the early phase of an oxidative stress response of macrophages upon phorbol 12-myristate 13-acetate (PMA) stimulation, allowing detailed analysis of the initial macrophage response upon a single biochemical stimulus within seconds after its application, thereby eliminating delay times introduced by other techniques during the stimulation procedure. Hence, an unprecedented view of the early kinetics of macrophage polarization is provided.

Aliphatic Quaternary Ammonium Functionalized Nanogels for Gene Delivery.

Gene therapy is a promising treatment for hereditary diseases, as well as acquired genetic diseases, including cancer. Facing the complicated physiological and pathological environment in vivo, developing efficient non-viral gene vectors is needed for their clinical application. Here, poly(N-isopropylacrylamide) (p(NIPAM)) nanogels are presented with either protonatable tertiary amine groups or permanently charged quaternized ammonium groups to achieve DNA complexation ability. In addition, a quaternary ammonium-functionalized nanogel was further provided with an aliphatic moiety using 1-bromododecane to add a membrane-interacting structure to ultimately facilitate intracellular release of the genetic material. The ability of the tertiary amine-, quaternized ammonium-, and aliphatic quaternized ammonium-functionalized p(NIPAM) nanogels (i.e., NGs, NGs-MI, and NGs-BDD, respectively) to mediate gene transfection was evaluated by fluorescence microscopy and flow cytometry. It is observed that NGs-BDD/pDNA complexes exhibit efficient gene loading, gene protection ability, and intracellular uptake similar to that of NGs-MI/pDNA complexes. However, only the NGs-BDD/pDNA complexes show a notable gene transfer efficiency, which can be ascribed to their ability to mediate DNA escape from endosomes. We conclude that NGs-BDD displays a cationic lipid-like behavior that facilitates endosomal escape by perturbing the endosomal/lysosomal membrane. These findings demonstrate that the presence of aliphatic chains within the nanogel is instrumental in accomplishing gene delivery, which provides a rationale for the further development of nanogel-based gene delivery systems.

3D-Printable Hierarchical Nanogel-GelMA Composite Hydrogel System.

The strength of the extracellular matrix (ECM) is that it is hierarchical in terms of matrix built-up, matrix density and fiber structure, which allows for hormones, cytokines, and other small biomolecules to be stored within its network. The ECM-like hydrogels that are currently used do not possess this ability, and long-term storage, along with the need for free diffusion of small molecules, are generally incompatible requirements. Nanogels are able to fulfill the additional requirements upon successful integration. Herein, a stable hierarchical nanogel-gelatin methacryloyl (GelMA) composite hydrogel system is provided by covalently embedding nanogels inside the micropore network of GelMA hydrogel to allow a controlled local functionality that is not found in a homogenous GelMA hydrogel. Nanogels have emerged as a powerful tool in nanomedicine and are highly versatile, due to their simplicity of chemical control and biological compatibility. In this study, an N-isopropylacrylamide-based nanogel with primary amine groups on the surface was modified with methacryloyl groups to obtain a photo-cross-linking ability similar to GelMA. The nanogel-GelMA composite hydrogel was formed by mixing the GelMA and the photo-initiator within the nanogel solution through UV irradiation. The morphology of the composite hydrogel was observed by scanning electron microscopy, which clearly showed the nanogel wrapped within the GelMA network and covering the surface of the pore wall. A release experiment was conducted to prove covalent bonding and the stability of the nanogel inside the GelMA hydrogel. In addition, 3D printability studies showed that the nanogel-GelMA composite ink is printable. Therefore, the suggested stable hierarchical nanogel-GelMA composite hydrogel system has great potential to achieve the in situ delivery and controllable release of bioactive molecules in 3D cell culture systems.

pH Sensitive Dextran Coated Fluorescent Nanodiamonds as a Biomarker for HeLa Cells Endocytic Pathway and Increased Cellular Uptake.

Fluorescent nanodiamonds are a useful for biosensing of intracellular signaling networks or environmental changes (such as temperature, pH or free radical generation). HeLa cells are interesting to study with these nanodiamonds since they are a model cell system that is widely used to study cancer-related diseases. However, they only internalize low numbers of nanodiamond particles very slowly via the endocytosis pathway. In this work, we show that pH-sensitive, dextran-coated fluorescent nanodiamonds can be used to visualise this pathway. Additionally, this coating improved diamond uptake in HeLa cells by 5.3 times (*** p < 0.0001) and decreased the required time for uptake to only 30 min. We demonstrated further that nanodiamonds enter HeLa cells via endolysosomes and are eventually expelled by cells.