Liquid Crystalline Network Microstructures for Stimuli Responsive Labels with Multi-Level Encryption.

Two-photon direct laser writing enables the fabrication of shape-changing microstructures that can be exploited in stimuli responsive micro-robotics and photonics. The use of Liquid Crystalline Networks (LCN) allows to realize 3D micrometric objects that can contract along a specific direction in response to stimuli, such as temperature or light. In this paper, the fabrication of free-standing LCN microstructures is demonstrated as graphical units of a smart tag for simple physical and optical encryption. Using an array of identical pixels, information can be hidden to the observer and revealed only upon application of a specific stimulus. The reading mechanism is based on the shape-change of each pixel under stimuli and their color that combine together in a two-level encryption label. Once the stimulus is removed, the pixels recover their original shape and the message remains completely hidden. Therefore, an opto-mechanical equivalent of an "invisible ink" is realized. This new concept paves the way for introducing enhanced functionalities in smart micro-systems within a single lithography step, spanning from storage devices with physical encryption to complex motion actuators.

Development of accessible platforms to promote myofibroblast differentiation by playing on hydrogel scaffold composition.

Mechanomimetic materials are particularly attractive for modeling in vitro fibroblast to myofibroblast (Myof) transition, a key process in the physiological repair of damaged tissue, and recognized as the core cellular mechanism of pathological fibrosis in different organs. In vivo, mechanical stimuli from the extracellular matrix (ECM) are crucial, together with cell-cell contacts and the pro-fibrotic transforming growth factor (TGF)-ß1, in promoting fibroblast differentiation. Here, we explore the impact of hydrogels made by polyacrylamide with different composition on fibroblast behavior. By appropriate modulation of the hydrogel composition (e.g. adjusting the crosslinker content), we produce and fully characterize three kinds of scaffolds with different Young modulus (E). We observe that soft hydrogels (E < 1 kPa) induced fibroblast differentiation better than stiffer ones, also in the absence of TGF-ß1. This study provides a readily accessible biomaterial platform to promote Myof generation. The easy approach used and the commercial availability of the monomers make these hydrogels suitable to a wide range of biomedical applications combined with high reproducibility and simple preparation protocols.

Corrigendum: Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: the impact of full-length dystrophin deficiency.

[This corrects the article DOI: 10.3389/fphys.2022.1030920.].

The Role of Crosslinker Molecular Structure on Mechanical and Light-Actuation Properties in Liquid Crystalline Networks.

Phase behavior modulation of liquid crystalline molecules can be addressed by structural modification at molecular level. Starting from a rigid rod-like core reduction of the symmetry or increase of the steric hindrance by different substituents generally reduces the clearing temperature. Similar approaches can be explored to modulate the properties of liquid crystalline networks (LCNs)-shape-changing materials employed as actuators in many fields. Depending on the application, the polymer properties have to be adjusted in terms of force developed under stimuli, kinetics of actuation, elasticity, and resistance to specific loads. In this work, the crosslinker modification at molecular level is explored towards the optimization of LCN properties as light-responsive artificial muscles. The synthesis and characterization of photopolymerizable crosslinkers, bearing different lateral groups on the aromatic core is reported. Such molecules are able to strongly modulate the material mechanical properties, such as kinetics and maximum tension under light actuation, opening up to interesting materials for biomedical applications.

Cellular Contact Guidance on Liquid Crystalline Networks with Anisotropic Roughness.

Cell contact guidance is widely employed to manipulate cell alignment and differentiation in vitro. The use of nano- or micro-patterned substrates allows efficient control of cell organization, thus opening up to biological models that cannot be reproduced spontaneously on standard culture dishes. In this paper, we explore the concept of cell contact guidance by Liquid Crystalline Networks (LCNs) presenting different surface topographies obtained by self-assembly of the monomeric mixture. The materials are prepared by photopolymerization of a low amount of diacrylate monomer dissolved in a liquid crystalline solvent, not participating in the reaction. The alignment of the liquid crystals, obtained before polymerization, determines the scaffold morphology, characterized by a nanometric structure. Such materials are able to drive the organization of different cell lines, e.g., fibroblasts and myoblasts, allowing for the alignment of single cells or high-density cell cultures. These results demonstrate the capabilities of rough surfaces prepared from the spontaneous assembly of liquid crystals to control biological models without the need of lithographic patterning or complex fabrication procedures. Interestingly, during myoblast differentiation, also myotube structuring in linear arrays is observed along the LCN fiber orientation. The implementation of this technology will open up to the formation of muscular tissue with well-aligned fibers in vitro mimicking the structure of native tissues.

Improvement of Hyperthermia Properties of Iron Oxide Nanoparticles by Surface Coating.

Magnetic hyperthermia is an oncological therapy that exploits magnetic nanoparticles activated by radiofrequency magnetic fields to produce a controlled temperature increase in a diseased tissue. The specific loss power (SLP) of magnetic nanoparticles or the capability to release heat can be improved using surface treatments, which can reduce agglomeration effects, thus impacting on local magnetostatic interactions. In this work, Fe3O4 nanoparticles are synthesized via a coprecipitation reaction and fully characterized in terms of structural, morphological, dimensional, magnetic, and hyperthermia properties (under the Hergt-Dutz limit). Different types of surface coatings are tested, comparing their impact on the heating efficacy and colloidal stability, resulting that sodium citrate leads to a doubling of the SLP with a substantial improvement in dispersion and stability in solution over time; an SLP value of around 170 W/g is obtained in this case for a 100 kHz and 48 kA/m magnetic field.

Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency.

Cardiomyocytes differentiated from human induced Pluripotent Stem Cells (hiPSC- CMs) are a unique source for modelling inherited cardiomyopathies. In particular, the possibility of observing maturation processes in a simple culture dish opens novel perspectives in the study of early-disease defects caused by genetic mutations before the onset of clinical manifestations. For instance, calcium handling abnormalities are considered as a leading cause of cardiomyocyte dysfunction in several genetic-based dilated cardiomyopathies, including rare types such as Duchenne Muscular Dystrophy (DMD)-associated cardiomyopathy. To better define the maturation of calcium handling we simultaneously measured action potential and calcium transients (Ca-Ts) using fluorescent indicators at specific time points. We combined micropatterned substrates with long-term cultures to improve maturation of hiPSC-CMs (60, 75 or 90 days post-differentiation). Control-(hiPSC)-CMs displayed increased maturation over time (90 vs 60 days), with longer action potential duration (APD), increased Ca-T amplitude, faster Ca-T rise (time to peak) and Ca-T decay (RT50). The progressively increased contribution of the SR to Ca release (estimated by post-rest potentiation or Caffeine-induced Ca-Ts) appeared as the main determinant of the progressive rise of Ca-T amplitude during maturation. As an example of severe cardiomyopathy with early onset, we compared hiPSC-CMs generated from a DMD patient (DMD-ΔExon50) and a CRISPR-Cas9 genome edited cell line isogenic to the healthy control with deletion of a G base at position 263 of the DMD gene (c.263delG-CMs). In DMD-hiPSC-CMs, changes of Ca-Ts during maturation were less pronounced: indeed, DMD cells at 90 days showed reduced Ca-T amplitude and faster Ca-T rise and RT50, as compared with control hiPSC-CMs. Caffeine-Ca-T was reduced in amplitude and had a slower time course, suggesting lower SR calcium content and NCX function in DMD vs control cells. Nonetheless, the inotropic and lusitropic responses to forskolin were preserved. CRISPR-induced c.263delG-CM line recapitulated the same developmental calcium handling alterations observed in DMD-CMs. We then tested the effects of micropatterned substrates with higher stiffness. In control hiPSC-CMs, higher stiffness leads to higher amplitude of Ca-T with faster decay kinetics. In hiPSC-CMs lacking full-length dystrophin, however, stiffer substrates did not modify Ca-Ts but only led to higher SR Ca content. These findings highlighted the inability of dystrophin-deficient cardiomyocytes to adjust their calcium homeostasis in response to increases of extracellular matrix stiffness, which suggests a mechanism occurring during the physiological and pathological development (i.e. fibrosis).

Women in Bioorganic Chemistry.

We are very happy to present this Special Issue, for which we acted as guest editors, and which includes scientific contributions from laboratories headed by women active in the field of bioorganic chemistry [...].

Novel Optics-Based Approaches for Cardiac Electrophysiology: A Review.

Optical techniques for recording and manipulating cellular electrophysiology have advanced rapidly in just a few decades. These developments allow for the analysis of cardiac cellular dynamics at multiple scales while largely overcoming the drawbacks associated with the use of electrodes. The recent advent of optogenetics opens up new possibilities for regional and tissue-level electrophysiological control and hold promise for future novel clinical applications. This article, which emerged from the international NOTICE workshop in 2018, reviews the state-of-the-art optical techniques used for cardiac electrophysiological research and the underlying biophysics. The design and performance of optical reporters and optogenetic actuators are reviewed along with limitations of current probes. The physics of light interaction with cardiac tissue is detailed and associated challenges with the use of optical sensors and actuators are presented. Case studies include the use of fluorescence recovery after photobleaching and super-resolution microscopy to explore the micro-structure of cardiac cells and a review of two photon and light sheet technologies applied to cardiac tissue. The emergence of cardiac optogenetics is reviewed and the current work exploring the potential clinical use of optogenetics is also described. Approaches which combine optogenetic manipulation and optical voltage measurement are discussed, in terms of platforms that allow real-time manipulation of whole heart electrophysiology in open and closed-loop systems to study optimal ways to terminate spiral arrhythmias. The design and operation of optics-based approaches that allow high-throughput cardiac electrophysiological assays is presented. Finally, emerging techniques of photo-acoustic imaging and stress sensors are described along with strategies for future development and establishment of these techniques in mainstream electrophysiological research.

Cell instructive Liquid Crystalline Networks for myotube formation.

Development of biological tissues in vitro is not a trivial task and requires the correct maturation of the selected cell line. To this aim, many attempts were done mainly by mimicking the biological environment using micro/nanopatterned or stimulated scaffolds. However, the obtainment of functional tissues in vitro is still far from being achieved. In contrast with the standard methods, we here present an easy approach for the maturation of myotubes toward the reproduction of muscular tissue. By using liquid crystalline networks with different stiffness and molecular alignment, we demonstrate how the material itself can give favorable interactions with myoblasts helping a correct differentiation. Electrophysiological studies demonstrate that myotubes obtained on these polymers have more adult-like morphology and better functional features with respect to those cultured on standard supports. The study opens to a platform for the differentiation of other cell lines in a simple and scalable way.

Photonic artificial muscles: from micro robots to tissue engineering.

Light responsive shape-changing polymers are able to mimic the function of biological muscles accomplishing mechanical work in response to selected stimuli. A variety of manufacturing techniques and chemical processes can be employed to shape these materials to different length scales, from centimeter fibers and films to 3D printed micrometric objects trying to replicate biological functions and operations. Controlled deformations shown to mimick basic animal operations such as walking, swimming or grabbing objects, while also controlling the refractive index and the geometry of devices, opens up the potential to implement tunable optical properties. Another possibility is that of combining artificial polymers with cells or biological tissue (such as intact cardiac trabeculae) with the aim to improve tissue formation in vitro or to support the mechanical function of damaged biological muscles. Such versatility is afforded by chemistry. New customized liquid crystalline monomers are presented here that modulate material properties for different applications. The role of synthetic material composition is highlighted as we demonstrate how using apparently similar molecular formulations, that liquid crystalline polymers can be adapted to different technological and medical challenges.

New Frontiers on Human Safe Insecticides and Fungicides: An Opinion on Trehalase Inhibitors.

In the era of green economy, trehalase inhibitors represent a valuable chance to develop non-toxic pesticides, being hydrophilic compounds that do not persist in the environment. The lesson on this topic that we learned from the past can be of great help in the research on new specific green pesticides. This review aims to describe the efforts made in the last 50 years in the evaluation of natural compounds and their analogues as trehalase inhibitors, in view of their potential use as insecticides and fungicides. Specifically, we analyzed trehalase inhibitors based on sugars and sugar mimics, focusing on those showing good inhibition properties towards insect trehalases. Despite their attractiveness as a target, up to now there are no trehalase inhibitors that have been developed as commercial insecticides. Although natural complex pseudo di- and trisaccharides were firstly studied to this aim, iminosugars look to be more promising, showing an excellent specificity profile towards insect trehalases. The results reported here represent an overview and a discussion of the best candidates which may lead to the development of an effective insecticide in the future.

Opposite Self-Folding Behavior of Polymeric Photoresponsive Actuators Enabled by a Molecular Approach.

The ability to obtain 3D polymeric objects by a 2D-to-3D shape-shifting method is very appealing for polymer integration with different materials, from metals in electronic devices to cells in biological studies. Such functional reshaping can be achieved through self-folding driven by a strain pattern designed into the molecular network. Among polymeric materials, liquid crystalline networks (LCNs) present an anisotropic molecular structure that can be exploited to tailor internal strain, resulting in a natural non-planar geometry when prepared in the form of flat films. In this article, we analyze the influence of different molecular parameters of the monomers on the spontaneous shape of the polymeric films and their deformation under different stimuli, such as heating or light irradiation. Modifying the alkilic chains of the crosslinkers is a simple and highly effective way to increase the temperature sensitivity of the final actuator, while modifying ester orientation on the aromatic core interestingly acts on the bending direction. Combining such effects, we have demonstrated that LCN stripes made of different monomeric mixtures originate complex non-symmetric deformation under light activation, thus opening up new applications in photonic and robotics.

Optical Investigation of Action Potential and Calcium Handling Maturation of hiPSC-Cardiomyocytes on Biomimetic Substrates.

Cardiomyocytes from human induced pluripotent stem cells (hiPSC-CMs) are the most promising human source with preserved genetic background of healthy individuals or patients. This study aimed to establish a systematic procedure for exploring development of hiPSC-CM functional output to predict genetic cardiomyopathy outcomes and identify molecular targets for therapy. Biomimetic substrates with microtopography and physiological stiffness can overcome the immaturity of hiPSC-CM function. We have developed a custom-made apparatus for simultaneous optical measurements of hiPSC-CM action potential and calcium transients to correlate these parameters at specific time points (day 60, 75 and 90 post differentiation) and under inotropic interventions. In later-stages, single hiPSC-CMs revealed prolonged action potential duration, increased calcium transient amplitude and shorter duration that closely resembled those of human adult cardiomyocytes from fresh ventricular tissue of patients. Thus, the major contribution of sarcoplasmic reticulum and positive inotropic response to ß-adrenergic stimulation are time-dependent events underlying excitation contraction coupling (ECC) maturation of hiPSC-CM; biomimetic substrates can promote calcium-handling regulation towards adult-like kinetics. Simultaneous optical recordings of long-term cultured hiPSC-CMs on biomimetic substrates favor high-throughput electrophysiological analysis aimed at testing (mechanistic hypothesis on) disease progression and pharmacological interventions in patient-derived hiPSC-CMs.

Development of Light-Responsive Liquid Crystalline Elastomers to Assist Cardiac Contraction.

RATIONALE: Despite major advances in cardiovascular medicine, heart disease remains a leading cause of death worldwide. However, the field of tissue engineering has been growing exponentially in the last decade and restoring heart functionality is now an affordable target; yet, new materials are still needed for effectively provide rapid and long-lasting interventions. Liquid crystalline elastomers (LCEs) are biocompatible polymers able to reversibly change shape in response to a given stimulus and generate movement. Once stimulated, LCEs can produce tension or movement like a muscle. However, so far their application in biology was limited by slow response times and a modest possibility to modulate tension levels during activation. OBJECTIVE: To develop suitable LCE-based materials to assist cardiac contraction. METHODS AND RESULTS: Thanks to a quick, simple, and versatile synthetic approach, a palette of biocompatible acrylate-based light-responsive LCEs with different molecular composition was prepared and mechanically characterized. Out of this, the more compliant one was selected. This material was able to contract for some weeks when activated with very low light intensity within a physiological environment. Its contraction was modulated in terms of light intensity, stimulation frequency, and ton/toff ratio to fit different contraction amplitude/time courses, including those of the human heart. Finally, LCE strips were mounted in parallel with cardiac trabeculae, and we demonstrated their ability to improve muscular systolic function, with no impact on diastolic properties. CONCLUSIONS: Our results indicated LCEs are promising in assisting cardiac mechanical function and developing a new generation of contraction assist devices.

Liquid Crystal-Induced Myoblast Alignment.

The ability to control cell alignment represents a fundamental requirement toward the production of tissue in vitro but also to create biohybrid materials presenting the functional properties of human organs. However, cell cultures on standard commercial supports do not provide a selective control on the cell organization morphology, and different techniques, such as the use of patterned or stimulated substrates, are developed to induce cellular alignment. In this work, a new approach toward in vitro muscular tissue morphogenesis is presented exploiting liquid crystalline networks. By using smooth polymeric films with planar homogeneous alignment, a certain degree of cellular order is observed in myoblast cultures with direction of higher cell alignment corresponding to the nematic director. The molecular organization inside the polymer determines such effects since no cell organization is observed using homeotropic or isotropic samples. These findings represent the first example of cellular alignment induced by the interaction with a nematic polymeric scaffold, setting the stage for new applications of liquid crystal polymers as active matter to control tissue growth.

Polarization-dependent deformation in light responsive polymers doped by dichroic dyes.

Light represents a very versatile stimulus and its use to control the deformation in shape-changing polymers can take advantage of multiple parameters (such as wavelength, intensity and polarization) to be explored in order to obtain differentiated responses. Polymers with selected color responsiveness are commonly prepared by using different dyes, while a polarization-dependent control can be introduced exploiting trans-cis isomerization of azobenzenes. As shape-changing polymers driven by a photothermal effect are gaining more and more attention in many application fields, exploring polarization to modulate their response could enlarge the tuning parameter space and provide an insight into the material optical properties. In this work, we demonstrate the effect of light polarization on the deformation of liquid crystalline networks doped by a small amount of a push-pull azobenzene. We demonstrate how enhancing the dye alignment in the polymeric matrix leads to different deformations by orthogonal polarizations. These results demonstrate polarization as a convenient further degree of freedom besides wavelength and intensity of the light stimulus.

Advances in Cell Scaffolds for Tissue Engineering: The Value of Liquid Crystalline Elastomers.

Recent discoveries evidenced that many cells organize into well-aligned nematic domains, showing also their topological defects and suggesting the liquid crystalline order to be necessary for some biological functions. These evidences were described as the basis for the development of a new area of research in which polymeric liquid crystals were developed to exploit and promote cell adhesion and proliferation towards tissue regeneration. To address the requirements of tissue engineering, new biocompatible materials must be designed and synthesized to support cell adherence and growth together with nutrient transport under physiological condition. This Minireview presents a journey that, starting from the first discovery of liquid crystalline phases in biological (natural) materials with different structures and physical-chemical properties, will inform readers of the very recent application of liquid crystal polymeric materials as functional cell scaffolds to address current tissue engineering issues.

Photonic Microhand with Autonomous Action.

Grabbing and holding objects at the microscale is a complex function, even for microscopic living animals. Inspired by the hominid-type hand, a microscopic equivalent able to catch microelements is engineered. This microhand is light sensitive and can be either remotely controlled by optical illumination or can act autonomously and grab small particles on the basis of their optical properties. Since the energy is delivered optically, without the need for wires or batteries, the artificial hand can be shrunk down to the micrometer scale. Soft material is used, in particular, a custom-made liquid-crystal network that is patterned by a photolithographic technique. The elastic reshaping properties of this material allow finger movement, using environmental light as the only energy source. The hand can be either controlled externally (via the light field), or else the conditions in which it autonomously grabs a particle in its vicinity can be created. This microrobot has the unique feature that it can distinguish between particles of different colors and gray levels. The realization of this autonomous hand constitutes a crucial element in the development of microscopic creatures that can perform tasks without human intervention and self-organized automation at the micrometer scale.

Liquid Crystalline Networks toward Regenerative Medicine and Tissue Repair.

The communication reports the use of liquid crystalline networks (LCNs) for engineering tissue cultures with human cells. Their ability as cell scaffolds for different cell lines is demonstrated. Preliminary assessments of the material biocompatibility are performed on human dermal fibroblasts and murine muscle cells (C2C12), demonstrating that coatings or other treatments are not needed to use the acrylate-based materials as support. Moreover, it is found that adherent C2C12 cells undergo differentiation, forming multinucleated myotubes, which show the typical elongated shape, and contain bundles of stress fibers. Once biocompatibility is demonstrated, the same LCN films are used as a substrate for culturing human induced pluripotent stem cell-derived cardiomyocites (hiPSC-CMs) proving that LCNs are capable to develop adult-like dimensions and a more mature cell function in a short period of culture in respect to standard supports. The demonstrated biocompatibility together with the extraordinary features of LCNs opens to preparation of complex cell scaffolds, both patterned and stimulated, for dynamic cell culturing. The ability of these materials to improve cell maturation and differentiation will be developed toward engineered heart and skeletal muscular tissues exploring regenerative medicine toward bioartificial muscles for injured sites replacement.