Mechanical Properties of Polydiacetylene Multilayers Studied by AFM Force Spectroscopy.

The blue-to-red chromatic phase transition of polydiacetylene (PDA) is accompanied by the twist and rearrangement of its side chains, which results in shortening of the conjugation length in the backbone. However, how these morphological changes affect its mechanical properties remains elusive. In this work, force spectroscopy mapping by atomic force microscopy was employed to quantify mechanical parameters of PDA thin films such as breakthrough force and Young's modulus at the monomer, blue, and red phases during the chromatic transition. We found that the breakthrough force increased by 113% and Young's modulus decreased by 21% during the blue-to-red transition, highlighting that the subtle change in the side-chain configuration has a dramatic impact on its mechanical properties.

Biomechanics of Macrophages on Disordered Surface Nanotopography.

Macrophages are involved in every stage of the innate/inflammatory immune responses in the body tissues, including the resolution of the reaction, and they do so in close collaboration with the extracellular matrix (ECM). Simplified substrates with nanotopographical features attempt to mimic the structural properties of the ECM to clarify the functional features of the interaction of the ECM with macrophages. We still have a limited understanding of the macrophage behavior upon interaction with disordered nanotopography, especially with features smaller than 10 nm. Here, we combine atomic force microscopy (AFM), finite element modeling (FEM), and quantitative biochemical approaches in order to understand the mechanotransduction from the nanostructured surface into cellular responses. AFM experiments show a decrease of macrophage stiffness, measured with the Young's modulus, as a biomechanical response to a nanostructured (ns-) ZrOx surface. FEM experiments suggest that ZrOx surfaces with increasing roughness represent weaker mechanical boundary conditions. The mechanical cues from the substrate are transduced into the cell through the formation of integrin-regulated focal adhesions and cytoskeletal reorganization, which, in turn, modulate cell biomechanics by downregulating cell stiffness. Surface nanotopography and consequent biomechanical response impact the overall behavior of macrophages by increasing movement and phagocytic ability without significantly influencing their inflammatory behavior. Our study suggests a strong potential of surface nanotopography for the regulation of macrophage functions, which implies a prospective application relative to coating technology for biomedical devices.

Deep learning strategy for small dataset from atomic force microscopy mechano-imaging on macrophages phenotypes.

The cytoskeleton is involved during movement, shaping, resilience, and functionality in immune system cells. Biomarkers such as elasticity and adhesion can be promising alternatives to detect the status of cells upon phenotype activation in correlation with functionality. For instance, professional immune cells such as macrophages undergo phenotype functional polarization, and their biomechanical behaviors can be used as indicators for early diagnostics. For this purpose, combining the biomechanical sensitivity of atomic force microscopy (AFM) with the automation and performance of a deep neural network (DNN) is a promising strategy to distinguish and classify different activation states. To resolve the issue of small datasets in AFM-typical experiments, nanomechanical maps were divided into pixels with additional localization data. On such an enlarged dataset, a DNN was trained by multimodal fusion, and the prediction was obtained by voting classification. Without using conventional biomarkers, our algorithm demonstrated high performance in predicting the phenotype of macrophages. Moreover, permutation feature importance was employed to interpret the results and unveil the importance of different biophysical properties and, in turn, correlated this with the local density of the cytoskeleton. While our results were demonstrated on the RAW264.7 model cell line, we expect that our methodology could be opportunely customized and applied to distinguish different cell systems and correlate feature importance with biophysical properties to unveil innovative markers for diagnostics.

Defined Physicochemical Cues Steering Direct Neuronal Reprogramming on Colloidal Self-Assembled Patterns (cSAPs).

Direct neuronal reprogramming of somatic cells into induced neurons (iNs) has been recently established as a promising approach to generating neuron cells. Previous studies have reported that the biophysical cues of the in vitro microenvironment are potent modulators in the cell fate decision; thus, the present study explores the effects of a customized pattern (named colloidal self-assembled patterns, cSAPs) on iN generation from human fibroblasts using small molecules. The result revealed that the cSAP, composed of binary particles in a hexagonal-close-packed (hcp) geometry, is capable of improving neuronal reprogramming efficiency and steering the ratio of the iN subtypes. Cells exhibited distinct cell morphology, upregulated cell adhesion markers (i.e., SDC1 and ITGAV), enriched signaling pathways (i.e., Hippo and Wnt), and chromatin remodeling on the cSAP compared to those on the control substrates. The result also showed that the iN subtype specification on cSAP was surface-dependent; therefore, the defined physicochemical cue from each cSAP is exclusive. Our findings show that direct cell reprogramming can be manipulated through specific biophysical cues on the artificial matrix, which is significant in cell transdifferentiation and lineage conversion.

Interaction of imidazolium-based ionic liquids with supported phospholipid bilayers as model biomembranes.

The cytotoxicity of ionic liquids (ILs) has been receiving attention in the context of the biological and environmental impact of their vast field of applications. It has been ascertained that the cell membrane is the main target of ILs when they interact with microorganisms, cells and bacteria; nevertheless, studies at the micro- and nano-scale aiming at better understanding of the fundamental mechanisms of toxicity of ILs are lacking. In this work, we used atomic force microscopy (AFM) to investigate the impact of room-temperature ILs on the mechanical, morphological and electrostatic properties of solid-supported DOPC phospholipid bilayers, taken as models of biomembranes. In particular, we have characterized the concentration-dependent and time-dependent evolution of the morphological, structural and mechanical properties of DOPC lipid membranes in the presence of imidazolium-based ILs with different alkyl chain lengths and hydrophilic/hydrophobic characteristics. The majority of ILs investigated were found to possess the ability of restructuring the lipid bilayer, through the formation of new IL/lipid complexes, showing distinctive morphological features (increase of area and roughness). The nanomechanical analysis of the lipid membrane exposed to ILs revealed a progressive, concentration-dependent perturbation of the structural ordering and rigidity of the membrane, evidenced by a decrease in the breakthrough force, Young's modulus and area stretching modulus. AFM detected a modification of the electrostatic double-layer at the membrane surface, in terms of a reduction of the original negative surface charge density, suggesting a progressive stratification of cations on the exposed leaflet of the lipid membrane. Our findings may be helpful in designing novel ILs with tailored interaction with biological membranes.

A study of macrophage mechanical properties and functional modulation based on the Young's modulus of PLGA-PEG fibers.

The immune response of macrophages plays an important role in defending against viral infection, tumor deterioration and repairing of contused tissue. Macrophage functional differentiation induced by nanodrugs is the leading edge of current research, but nanodrugs have toxic side effects, and the influence of their physical properties on macrophages is not clear. Here we create an alternative way to modulate macrophage function through PLGA-PEG fibers' Young's modulus. Previously, we revealed that by controlling the Young's modulus of the fibers from kPa to MPa, all the fibers entered murine macrophage cells (RWA 264.7) in a similar manner, and based on that, we found that macrophages' mechanical properties were affected by the fibers' Young's modulus, that is, hard fibers with a Young's modulus of ∼1 MPa increased the cell average Young's modulus, but did not affect the cell shape, while soft fibers with a Young's modulus of ∼100 kPa decreased the cell average Young's modulus and modulated the cell shape to a more spherical one. On the other hand, only the soft fibers induced proinflammatory cytokine secretion, indicating an M1 macrophage functional modulation by low Young's modulus fibers. This study explored the mechanical properties of the interactions between PLGA-PEG fibers and cells, in particular, when guiding the direction of the modulation of macrophage function, which is of great significance for the applications of material biology in the biomedical field.

Polydopamine-Mediated Protein Adsorption Alters the Epigenetic Status and Differentiation of Primary Human Adipose-Derived Stem Cells (hASCs).

Polydopamine (PDA) is a biocompatible cell-adhesive polymer with versatile applications in biomedical devices. Previous studies have shown that PDA coating could improve cell adhesion and differentiation of human mesenchymal stem cells (hMSCs). However, there is still a knowledge gap in the effect of PDA-mediated protein adsorption on the epigenetic status of MSCs. This work used gelatin-coated cell culture surfaces with and without PDA underlayer (Gel and PDA-Gel) to culture and differentiate primary human adipose-derived stem cells (hASCs). The properties of these two substrates were significantly different, which, in combination with a variation in extracellular matrix (ECM) protein bioactivity, regulated cell adhesion and migration. hASCs reduced focal adhesions by downregulating the expression of integrins such as αV, α1, α2, and ß1 on the PDA-Gel compared to the Gel substrate. Interestingly, the ratio of H3K27me3 to H3K27me3+H3K4me3 was decreased, but this only occurred for upregulation of AGG and BMP4 genes during chondrogenic differentiation. This result implies that the PDA-Gel surface positively affects the chondrogenic, but not adipogenic and osteogenic, differentiation. In conclusion, for the first time, this study demonstrates the sequential effects of PDA coating on the biophysical property of adsorbed protein and then focal adhesions and differentiation of hMSCs through epigenetic regulation. This study sheds light on PDA-mediated mechanotransduction.

A biomimetic hyaluronic acid-silk fibroin nanofiber scaffold promoting regeneration of transected urothelium.

This study was designed to investigate the regulatory effect of hyaluronic acid (HA)-coating silk fibroin (SF) nanofibers during epithelialization of urinary tract for urethral regeneration. The obtained electrospun biomimetic tubular HA-SF nanofiber scaffold is composed of a dense inner layer and a porous outer layer in order to mimic adhesion and cavernous layers of the native tissue, respectively. A thin layer of HA-gel coating was fixed in the inner wall to provide SF nanofibers with a dense and smooth surface nano-topography and higher hydrophilicity. Compared with pure SF nanofibers, HA-SF nanofibers significantly promoted the adhesion, growth, and proliferation of primary urothelial cells, and up-regulate the expression of uroplakin-3 (terminal differentiation keratin protein in urothelium). Using the New Zealand male rabbit urethral injury model, the scaffold composed of tubular HA-SF nanofibers could recruit lumen and myoepithelial cells from the adjacent area of the host, rapidly reconstructing the urothelial barrier in the wound area in order to keep the urinary tract unobstructed, thereby promoting luminal epithelialization, smooth muscle bundle structural remodeling, and capillary formation. Overall, the synergistic effects of nano-topography and biophysical cues in a biomimetic scaffold design for effective endogenous regeneration.

Molybdenum Diphosphide Nanorods with Laser-Potentiated Peroxidase Catalytic/Mild-Photothermal Therapy of Oral Cancer.

Chemodynamic therapy (CDT) is an emerging treatment that usually employs chemical agents to decompose hydrogen peroxide (H2O2) into hydroxyl radical (•OH) via Fenton or Fenton-like reactions, inducing cell apoptosis or necrosis by damaging biomacromolecules such as, lipids, proteins, and DNA. Generally, CDT shows high tumor-specificity and minimal-invasiveness in patients, thus it has attracted extensive research interests. However, the catalytic reaction efficiency of CDT is largely limited by the relatively high pH at the tumor sites. Herein, a 808 nm laser-potentiated peroxidase catalytic/mild-photothermal therapy of molybdenum diphosphide nanorods (MoP2 NRs) is developed to improve CDT performance, and simultaneously achieve effective tumor eradication and anti-infection. In this system, MoP2 NRs exhibit a favorable cytocompatibility due to their inherent excellent elemental biocompatibility. Upon irradiation with an 808 nm laser, MoP2 NRs act as photosensitizers to efficiently capture the photo-excited band electrons and valance band holes, exhibiting enhanced peroxidase-like catalytic activity to sustainedly decompose tumor endogenous H2O2 to •OH, which subsequently destroy the cellular biomacromolecules both in tumor cells and bacteria. As demonstrated both in vitro and in vivo, this system exhibits a superior therapeutic efficiency with inappreciable toxicity. Hence, the work may provide a promising therapeutic technique for further clinical applications.

In vivo performance of electrospun tubular hyaluronic acid/collagen nanofibrous scaffolds for vascular reconstruction in the rabbit model.

One of the main challenges of tissue-engineered vascular prostheses is restenosis due to intimal hyperplasia. The aim of this study is to develop a material for scaffolds able to support cell growth while tolerating physiological conditions and maintaining the patency of carotid artery model. Tubular hyaluronic acid (HA)-functionalized collagen nanofibrous composite scaffolds were prepared by sequential electrospinning method. The tubular composite scaffold has well-controlled biophysical and biochemical signals, providing a good matrix for the adhesion and proliferation of vascular endothelial cells (ECs), but resisting to platelets adhesion when exposed to blood. Carotid artery replacement experiment from 6-week rabbits showed that the HA/collagen nanofibrous composite scaffold grafts with endothelialization on the luminal surface could maintain vascular patency. At retrieval, the composite scaffold maintained good structural integrity and had comparable mechanical strength as the native artery. This study indicating that electrospun scaffolds combined with cells may become an alternative to prosthetic grafts for vascular reconstruction.

Hyaluronic Acid/Collagen Nanofiber Tubular Scaffolds Support Endothelial Cell Proliferation, Phenotypic Shape and Endothelialization.

In this study, we designed and synthetized artificial vascular scaffolds based on nanofibers of collagen functionalized with hyaluronic acid (HA) in order to direct the phenotypic shape, proliferation, and complete endothelization of mouse primary aortic endothelial cells (PAECs). Layered tubular HA/collagen nanofibers were prepared using electrospinning and crosslinking process. The obtained scaffold is composed of a thin inner layer and a thick outer layer that structurally mimic the layer the intima and media layers of the native blood vessels, respectively. Compared with the pure tubular collagen nanofibers, the surface of HA functionalized collagen nanofibers has higher anisotropic wettability and mechanical flexibility. HA/collagen nanofibers can significantly promote the elongation, proliferation and phenotypic shape expression of PAECs. In vitro co-culture of mouse PAECs and their corresponding smooth muscle cells (SMCs) showed that the luminal endothelialization governs the biophysical integrity of the newly formed extracellular matrix (e.g., collagen and elastin fibers) and structural remodeling of SMCs. Furthermore, in vitro hemocompatibility assays indicated that HA/collagen nanofibers have no detectable degree of hemolysis and coagulation, suggesting their promise as engineered vascular implants.

Synthetic preparations and atomic scale engineering of silver nanoparticles for biomedical applications.

Owing to their peculiar oxidative effect, silver cations (Ag+) are well known for their antimicrobial properties and explored as therapeutic agents for biomedical applications. Size control with improved dispersion and stability are the key factors of Ag NPs (silver nanoparticles) to be used in biomedical applications. Silver based nano-materials are highly efficient due to their biological, chemical and physical properties in comparison with bulk silver. Atomic scale fabrication is achieved by rearranging the internal components of a material, in turn, influencing the mechanical, electrical, magnetic, thermal and chemical properties. For instance, size and shape have a strong impact on the optical, thermal and catalytic properties of Ag NPs. Such properties can be tuned by controlling the surface/volume ratio of Ag nanostructures with a small size (ideally <100 nm), in turn showing peculiar biological activity different from that of bulk silver. Silver nanomaterials such as nanoparticles, thin films and nanorods can be synthesized by various physical, chemical and biological methods whose most recent implementations will be described in this review. By controlling the structure-functionality relationship, silver based nano-materials have high potential for commercialization in biomedical applications. Antimicrobial, antifungal, antiviral, and anti-inflammatory Ag NPs can be applied in several fields such as pharmaceutics, sensors, coatings, cosmetics, wound healing, bio-labelling agents, antiviral drugs, and packaging.

Hyaluronic acid-functionalized poly-lactic acid (PLA) microfibers regulate vascular endothelial cell proliferation and phenotypic shape expression.

This work was designed to evaluate the efficacy of hyaluronic acid (HA) functionalized tubular poly-lactic acid (PLA) microfibers in directing the luminal pre-endothelialization of vascular endothelial cells (ECs). Tubular HA/PLA microfibers with hierarchical architecture were prepared by electrospinning and chemical cross-linking process. A layer of HA microfibrous film coating was fixed on the inner wall surface of the tubular HA/PLA microfibers, resulting in higher anisotropy wettability and relatively lower surface energy and roughness. We confirmed that HA coating on PLA microfibers surface have reduced hemolytic activity and coagulation degree. Mouse vascular ECs exhibited surface-dependent differences in cell elongation and proliferation (HA/PLA > PLA). Compared with PLA microfibers, the gene expression levels of platelet EC adhesion molecule-1 (PECAM-1/CD31) and vascular endothelial growth factor (VEGF) in ECs of HA/PLA microfibers surface were up-regulated. Immunostaining analysis revealed that the surface of HA/PLA nanofibers supported the expression of mature vascular EC phenotype CD31 protein. In vitro co-culture analysis showed that the luminal pre-endothelialization induced vascular smooth muscle cells (SMCs) to maintain their phenotypic shape and establish natural behavior patterns in the hierarchical tubular scaffold. These studies indicate that the biophysical cues of scaffolds are potent regulators of vascular EC endothelialization.

Editing the Shape Morphing of Monocomponent Natural Polysaccharide Hydrogel Films.

Shape-morphing hydrogels can be widely used to develop artificial muscles, reconfigurable biodevices, and soft robotics. However, conventional approaches for developing shape-morphing hydrogels highly rely on composite materials or complex manufacturing techniques, which limit their practical applications. Herein, we develop an unprecedented strategy to edit the shape morphing of monocomponent natural polysaccharide hydrogel films via integrating gradient cross-linking density and geometry effect. Owing to the synergistic effect, the shape morphing of chitosan (CS) hydrogel films with gradient cross-linking density can be facilely edited by changing their geometries (length-to-width ratios or thicknesses). Therefore, helix, short-side rolling, and long-side rolling can be easily customized. Furthermore, various complex artificial 3D deformations such as artificial claw, horn, and flower can also be obtained by combining various flat CS hydrogel films with different geometries into one system, which can further demonstrate various shape transformations as triggered by pH. This work offers a simple strategy to construct a monocomponent hydrogel with geometry-directing programmable deformations, which provides universal insights into the design of shape-morphing polymers and will promote their applications in biodevices and soft robotics.

Rapid detection of SARS-CoV-2 viral nucleic acids based on surface enhanced infrared absorption spectroscopy.

Efficient point-of-care diagnosis of severe acute respiratory syndrome-corovavirus-2 (SARS-CoV-2) is crucial for the early control of novel coronavirus infections. At present, polymerase chain reaction (PCR) is primarily used to detect SARS-CoV-2. Despite the high sensitivity, the PCR process is time-consuming and complex which limits its applicability for rapid testing of large-scale outbreaks. Here, we propose a rapid and easy-to-implement approach for SARS-CoV-2 detection based on surface enhanced infrared absorption (SEIRA) spectroscopy. The evaporated gold nano-island films are used as SEIRA substrates which are functionalized with the single-stranded DNA probes for specific binding to selected SARS-CoV-2 genomic sequences. The infrared absorption spectra are analyzed using the principal component analysis method to identify the key characteristic differences between infected and control samples. The SEIRA-based biosensor demonstrates rapid detection of SARS-CoV-2, completing the detection of 1 µM viral nucleic acids within less than 5 min without any amplification. When combined with the recombinase polymerase amplification treatment, the detection capability of 2.98 copies per µL (5 aM) can be completed within 30 min. This approach provides a simple and economical alternative for COVID-19 diagnosis, which can be potentially useful in monitoring and controlling future pandemics in a timely manner.

Unveiling a Hidden Event in Fluorescence Correlative Microscopy by AFM Nanomechanical Analysis.

Fluorescent imaging combined with atomic force microscopy (AFM), namely AFM-fluorescence correlative microscopy, is a popular technology in life science. However, the influence of involved fluorophores on obtained mechanical information is normally underestimated, and such subtle changes are still challenging to detect. Herein, we combined AFM with laser light excitation to perform a mechanical quantitative analysis of a model membrane system labeled with a commonly used fluorophore. Mechanical quantification was additionally validated by finite element simulations. Upon staining, we noticed fluorophores forming a diffuse weakly organized overlayer on phospholipid supported membrane, easily detected by AFM mechanics. The laser was found to cause a degradation of mechanical stability of the membrane synergically with presence of fluorophore. In particular, a 30 min laser irradiation, with intensity similar to that in typical confocal scanning microscopy experiment, was found to result in a ∼40% decrease in the breakthrough force of the stained phospholipid bilayer along with a ∼30% reduction in its apparent elastic modulus. The findings highlight the significance of analytical power provided by AFM, which will allow us to "see" the "unseen" in correlative microscopy, as well as the necessity to consider photothermal effects when using fluorescent dyes to investigate, for example, the deformability and permeability of phospholipid membranes.

An amphiphilic aggregate-induced emission polyurethane probe for in situ actin observation in living cells.

The specific binding of fluorescent probes or biomolecules to the actin cytoskeleton network is increasingly important for monitoring various complex cellular activities such as cell adhesion, proliferation, locomotion, endocytosis, and cell division. However, improving cell uptake and subcellular resolution is still the main obstacle for successful and wide application of cellular fluorescent probes. Here, we designed and synthesized an amphiphilic block polyurethane with peculiar photophysical properties of aggregation induced emission (AIE), which can be used in living cell imaging to promote selective visualization of cell structures. The AIE effect polyurethane (abbreviated as AIE-PU) was prepared by two-step polymerization of diisocyanate terminated polyethylene glycol and polycaprolactone with hydroxyl terminated AIE dye. A series of characterization techniques proved the successful synthesis of AIE-PU. Due to the amphiphilic chain segment of its linear block molecule, AIE-PU block copolymers can self-assemble into spherical nanoparticles in aqueous solution, showing relatively stable photophysical properties and good water dispersion. Cellular experiments demonstrated that AIE-PUs have low toxicity and high actin network affinity. Moreover, the uptake mechanism was studied by low temperature and metabolic inhibition experiments, showing that AIE-PU nanoparticles could be easily internalized into different living cells through energy-dependent endocytosis, and can be transported from the cellular periphery to the actin network via clathrin- and caveolae-dependent transport pathway. Upon binding with the actin network, the inter-chain AIE mechanism of the probe was significantly enhanced, which is pivotal for the long-term stable fluorescence imaging of actin microfilament network in living cells. Finally, compared with commercial actin dyes, this probe showed higher photostability, even after a longer retention time, without significant fluorescence quenching.

A biodegradable block polyurethane nerve-guidance scaffold enhancing rapid vascularization and promoting reconstruction of transected sciatic nerve in Sprague-Dawley rats.

Reconstruction of peripheral nerve defects with tissue engineered nerve scaffolds is an exciting field of biomedical research and holds potential for clinical application. However, due to poor neovascularization after the implantation, nerve regeneration is still not satisfactory, especially for large nerve defects. These obstacles hinder the investigation of basic neurobiological principles and development of a wide range of treatments for peripheral nerve diseases. Herein, we designed an amphiphilic alternating block polyurethane (abbreviated as PU) copolymer-based nerve guidance scaffold, which has good Schwann cell compatibility, and more importantly, a rapid vascularization of the scaffold in vivo. In the sciatic nerve transection model of SD rats, vascularized PU nerve guidance scaffolds induced rapid regeneration of nerve fibers and axons along the scaffold. Through the analysis of nerve electrophysiology, sciatic nerve functional index, histology, and immunofluorescence related to angiogenesis, we determined that PU with rapid vascularization function enhances recovery and re-obtains nerve conduction function. Our study points out a new strategy of using nerve tissue engineering scaffolds to treat large nerve defects.

Spatially Resolved Correlation between Stiffness Increase and Actin Aggregation around Nanofibers Internalized in Living Macrophages.

Plasticity and functional diversity of macrophages play an important role in resisting pathogens invasion, tumor progression and tissue repair. At present, nanodrug formulations are becoming increasingly important to induce and control the functional diversity of macrophages. In this framework, the internalization process of nanodrugs is co-regulated by a complex interplay of biochemistry, cell physiology and cell mechanics. From a biophysical perspective, little is known about cellular mechanics' modulation induced by the nanodrug carrier's internalization. In this study, we used the polylactic-co-glycolic acid (PLGA)-polyethylene glycol (PEG) nanofibers as a model drug carrier, and we investigated their influence on macrophage mechanics. Interestingly, the nanofibers internalized in macrophages induced a local increase of stiffness detected by atomic force microscopy (AFM) nanomechanical investigation. Confocal laser scanning microscopy revealed a thickening of actin filaments around nanofibers during the internalization process. Following geometry and mechanical properties by AFM, indentation experiments are virtualized in a finite element model simulation. It turned out that it is necessary to include an additional actin wrapping layer around nanofiber in order to achieve similar reaction force of AFM experiments, consistent with confocal observation. The quantitative investigation of actin reconfiguration around internalized nanofibers can be exploited to develop novel strategies for drug delivery.