Cardiotoxicity Assessment through a Polymer-Based Cantilever Platform: An Integrated Electro-Mechanical Screening Approach.

Preclinical drug screening for cardiac toxicity has traditionally relied on observing changes in cardiomyocytes' electrical activity, primarily through invasive patch clamp techniques or non-invasive microelectrode arrays (MEA). However, relying solely on field potential duration (FPD) measurements for electrophysiological assessment can miss the full spectrum of drug-induced toxicity, as different drugs affect cardiomyocytes through various mechanisms. A more comprehensive approach, combining field potential and contractility measurements, is essential for accurate toxicity profiling, particularly for drugs targeting contractile proteins without affecting electrophysiology. However, previously proposed platform has significant limitations in terms of simultaneous measurement. The novel platform addresses these issues, offering enhanced, non-invasive evaluation of drug-induced cardiotoxicity. It features eight cantilevers with patterned strain sensors and MEA, enabling real-time monitoring of both cardiomyocyte contraction force and field potential. This system can detect minimum cardiac contraction force of ≈2 µN and field potential signals with 50 µm MEA diameter, using the same cardiomyocytes in measurements of two parameters. Testing with six drugs of varied mechanisms of action, the platform successfully identifies these mechanisms and accurately assesses toxicity profiles, including drugs not inhibiting potassium channels. This innovative approach presents a comprehensive, non-invasive method for cardiac function assessment, poised to revolutionize preclinical cardiotoxicity screening.

Correction: Quantitative assessment of cardiomyocyte mechanobiology through high-throughput cantilever-based functional well plate systems.

Correction for 'Quantitative assessment of cardiomyocyte mechanobiology through high-throughput cantilever-based functional well plate systems' by Jongyun Kim et al., Analyst, 2023, 148, 5133-5143, https://doi.org/10.1039/D3AN01286G.

Correction: Enhanced cardiomyocyte structural and functional anisotropy through synergetic combination of topographical, conductive, and mechanical stimulation.

Correction for 'Enhanced cardiomyocyte structural and functional anisotropy through synergetic combination of topographical, conductive, and mechanical stimulation' by Jongyun Kim et al., Lab Chip, 2023, 23, 4540-4551, https://doi.org/10.1039/D3LC00451A.

Enhanced cardiomyocyte structural and functional anisotropy through synergetic combination of topographical, conductive, and mechanical stimulation.

Drug-induced cardiotoxicity, a significant concern in the pharmaceutical industry, often results in the withdrawal of drugs from the market. The main cause of drug-induced cardiotoxicity is the use of immature cardiomyocytes during in vitro drug screening procedures. Over time, several methods such as topographical, conductive, and mechanical stimulation have been proposed to enhance both maturation and contractile properties of these cardiomyocytes. However, the synergistic effects of integrating topographical, conductive, and mechanical stimulation for cardiomyocyte maturation remain underexplored and poorly understood. To address this limitation, herein, we propose a grooved polydimethylsiloxane (PDMS) membrane embedded with silver nanowires (AgNWs-E-PDMS). The proposed AgNWs-E-PDMS membrane enhances the maturation of cardiomyocytes and provides a more accurate evaluation of drug-induced cardiotoxicity. When subjected to 10% tensile stress on the AgNWs-E-PDMS membrane, cardiomyocytes displayed substantial enhancements. Specifically, the contraction force, sarcomere length, and connexin-43 (Cx43) expression are increased by 2.0-, 1.5-, and 2.4-times, respectively, compared to the control state. The practical feasibility of the proposed device as a drug screening platform is demonstrated by assessing the adverse effects of lidocaine on cardiomyocytes. The contraction force and beat rate of lidocaine treated cardiomyocytes cultured on the AgNWs-E-PDMS membrane under mechanical stimulation decreased to 0.9 and 0.64 times their initial values respectively, compared to 0.6 and 0.51 times in the control state. These less pronounced changes in the contraction force and beat rate signify the superior drug response in the cardiomyocytes, a result of their enhanced maturation and growth on the AgNWs-E-PDMS membrane combined with mechanical stimulation.

Quantitative assessment of cardiomyocyte mechanobiology through high-throughput cantilever-based functional well plate systems.

Proper regulation of the in vitro cell culture environment is essential for disease modelling and drug toxicity screening. The main limitation of well plates used for cell culture is that they cannot accurately maintain energy sources and compounds needed during cell growth. Herein, to understand the importance of perfusion in cardiomyocyte culture, changes in contractile force and heart rate during cardiomyocyte growth are systematically investigated, and the results are compared with those of a perfusion-free system. The proposed perfusion system consists of a Peltier refrigerator, a peristaltic pump, and a functional well plate. A functional well plate with 12 wells is made through injection moulding, with two tubes integrated in the cover for each well to continuously circulate the culture medium. The contractile force of cardiomyocytes growing on the cantilever surface is analysed through changes in cantilever displacement. The maturation of cardiomyocytes is evaluated through fluorescence staining and western blot; cardiomyocytes cultured in the perfusion system show greater maturity than those cultured in a manually replaced culture medium. The pH of the culture medium manually replaced at intervals of 3 days decreases to 6.8, resulting in an abnormal heartbeat, while cardiomyocytes cultured in the perfusion system maintained at pH 7.4 show improved contractility and a uniform heart rate. Two well-known ion channel blockers, verapamil and quinidine, are used to measure changes in the contractile force of cardiomyocytes from the two systems. Cardiomyocytes in the perfusion system show greater stability during drug toxicity screening, proving that the perfusion system provides a better environment for cell growth.

Efficient removal of methylene blue from aqueous solution by ZIF-8-decorated helicoidal electrospun polymer strips.

Immobilization of metal-organic frameworks (MOFs) on electrospun products for wastewater treatment has garnered considerable attention in recent years. However, the effect of the overall geometry and surface-area-to-volume ratio of MOF-decorated electrospun architectures on their performances have rarely been investigated. Herein, we prepared polycaprolactone (PCL)/polyvinylpyrrolidone (PVP) strips with helicoidal geometries via immersion electrospinning. By regulating the weight ratio of PCL to PVP, the morphologies and surface-area-to-volume ratios of the PCL/PVP strips could be controlled precisely. Then, the zeolitic imidazolate framework-8 (ZIF-8) for removing methylene blue (MB) from an aqueous solution was immobilized on the electrospun strips, resulting in ZIF-8-decorated PCL/PVP strips. The key characteristics of these composite products, such as adsorption and photocatalytic degradation behavior toward MB in the aqueous solution, were carefully investigated. Owing to the desired overall geometry and high surface-area-to-volume ratio of the ZIF-8-decorated helicoidal strips, a high MB adsorption capacity of 151.6 mg g-1 was obtained, which is significantly higher than those with conventional electrospun straight fibers. In addition, higher MB uptake rates, higher recycling and kinetic adsorption efficiencies, higher MB photocatalytic degradation efficiencies, and faster MB photocatalytic degradation rates were confirmed. This work provides new insights to improve the performance of existing and potential electrospun product-based water treatment strategies.

Multifunctional nanocomposite based on polyvinyl alcohol, cellulose nanocrystals, titanium dioxide, and apple peel extract for food packaging.

Although polyvinyl alcohol (PVA) is a potential biodegradable food packaging material, it has several critical limitations: low mechanical strength, poor ultraviolet (UV) and water vapor barrier properties, and lack of antioxidant and antimicrobial properties. Previous studies have used cellulose nanocrystals (CNCs) to improve the mechanical and water vapor barrier properties of the PVA matrix. In this study, a multifunctional nanocomposite for food packaging applications was developed by incorporating titanium dioxide (TiO2) and apple peel extract (APE) into a PVA/CNC matrix. The combination of TiO2 and APE in the nanocomposites not only enhanced multifunctionality but also improved mechanical and barrier properties. The mechanical strength and water vapor barrier properties of PVA/CNC/TiO2/APE (5 wt% TiO2 and 20 wt% APE in the PVA/CNC matrix containing 5 wt% of CNCs) increased by 49.9 % and 36.6 % compared to PVA. Furthermore, PVA/CNC/TiO2/APE exhibited an excellent UV barrier (UV-protection factor of 1012.73) and high antioxidant and antimicrobial properties. In food packaging tests with fresh cherry tomatoes and potatoes, PVA/CNC/TiO2/APE effectively protected samples from external influences and prolonged their self-life, demonstrating the potential use of this nanocomposite as a biodegradable and multifunctional food packaging material.

PVA/CNC/TiO2 nanocomposite for food-packaging: Improved mechanical, UV/water vapor barrier, and antimicrobial properties.

Although polyvinyl alcohol (PVA) is a promising biodegradable packaging material, it presents some disadvantages for food packaging such as poor ultraviolet (UV) and water vapor barrier properties, low mechanical strength, poor water resistance, and lack of antimicrobial properties. To overcome these limitations, novel PVA/cellulose nanocrystals (CNC)/titanium dioxide (TiO2) nanocomposites were developed, characterized, and demonstrated for potential food packaging applications. The mechanical strength, water vapor barrier, and UV barrier properties of PVA/CNC/TiO2 5 % film (5 wt% TiO2 in the PVA/CNC matrix with 5 wt% of CNCs) increased by 55.8 %, 45.2 %, and 70,056.8 %, respectively, compared to those of a PVA film. In the antibacterial simulation test, PVA/CNC/TiO2 5 % film could limit the growth of microorganisms for 14 days. In packaging tests with fresh garlic, PVA/CNC/TiO2 films effectively prevented weight loss and spoilage by external influences, indicating the potential of the PVA/CNC/TiO2 nanocomposites for food-packaging applications.

MEA-integrated cantilever platform for comparison of real-time change in electrophysiology and contractility of cardiomyocytes to drugs.

Drug-induced cardiotoxicity is a potentially severe side effect that can alter the contractility and electrophysiology of the cardiomyocytes. Cardiotoxicity is generally assessed through animal models using conventional drug screening platforms. Despite significant developments in drug screening platforms, the difficulty in measuring electrophysiology and contractile profile together affects the investigation of cardiotoxicity in potential drugs. Some drugs can prove to be more toxic to contractility than electrophysiology, which demands the need for a reliable, dual, and simultaneous drug screening platform. Herein, we propose the microelectrode array integrated SU-8 cantilever for dual and simultaneous measurement of electrophysiology and contractility of cardiomyocytes. The SU-8 cantilever is integrated with microelectrode array (C-MEA) using conventional photolithographic techniques. Drug tests are conducted to verify the feasibility of the C-MEA platform using three cardiovascular drugs. Clinically recognized drugs, quinidine and verapamil, are used to activate both the hERG channel and the contractile characteristics of cardiomyocytes. The effect of ion channel blockers on the field potential duration (FPD) of the cardiomyocytes is compared with several contractility-based parameters. The contraction-relaxation duration (CRD) profile is relatively close to that of FPD in tested drugs (half-maximal (IC50) toxicities are 1.093 µM (FPD) and 1.924 µM (CRD) for quinidine and 166.2 nM (FPD) and 459.4 nM (CRD) for verapamil). Blebbistatin, a known myosin II inhibitor, primarily affects the contractile profile of cardiomyocytes but not their field potential, with no evident correlation between contractility and field potential profiles. The proposed cantilever-based mechano-electrophysiology measurements platform provides a promising and accurate means to assess cardiotoxicity.

Facile generation of crumpled polymer strips by immersion electrospinning for oil spill cleanups.

The fabrication of electrospun products with desired structures and improved oil sorption capacities has attracted increasing attention in recent years; however, the overall geometry of electrospun products has rarely been investigated. Herein, we report a promising and competitive strategy for the preparation of novel electrospun polystyrene (PS) strips with advanced crumpled geometries for oil spill cleanups. The crumpled PS strips were prepared by immersion electrospinning, in which the nozzle and collector were simultaneously immersed in a coagulation bath. By controlling the processing parameters such as applied voltage and polymer concentration, the geometric morphology of the crumpled strips was precisely regulated. Benefiting from the crumpled structure, the crumpled strips demonstrated oil sorption capacities of approximately 83, 77, and 72 g/g for silicone, canola, and mineral oils, respectively. These values significantly higher than the values exhibited by the straight conventional electrospun fibers. The reported strategy offers a novel perspective on the rational design of electrospun products for oil sorption applications.

Role of heat treatment in improving replication quality of PDMS double-casting.

An efficient and eco-friendly method utilizing the heat treatment of the PDMS master is proposed for improving the replication quality of PDMS double-casting. The effects of heat treatment on interfacial adhesion are investigated in terms of uncured low molecular weight chains, surface energy, and surface roughness. The PDMS master treated at 150 °C for 72 h shows the highest replication quality of micropatterns with a diameter and height of 30 µm.

Polyvinyl alcohol/cellulose nanocrystals/alkyl ketene dimer nanocomposite as a novel biodegradable food packing material.

Polyvinyl alcohol (PVA) is used in many applications because of its excellent physicochemical properties, non-toxicity, and biodegradability. However, its relatively low water resistance, poor water vapor/ultraviolet (UV) barrier properties, and poor mechanical properties compared with conventional polymers limit its applications in food packaging. In this study, cellulose nanocrystals (CNCs) and alkyl ketene dimer (AKD) were used to overcome these issues. The mechanical properties, water resistance, and barrier properties of the developed PVA/CNC/AKD films were significantly improved relative to those of a neat PVA film. The mechanical strength of a PVA/CNC/AKD 15% film (15 wt% AKD in a PVA/CNC matrix of 5 wt% CNCs) was 64.6% and 37% higher than those of PVA and PVA/CNC films, respectively. The water vapor transmission rate, water absorption, and solubility of PVA/CNC/AKD 15% were 41.2%, 61.1%, and 92.9%, respectively (lower than those of the neat PVA film). In addition, the UV barrier properties and soil degradation of the PVA/CNC/AKD films were significantly improved.

The effect of topographical and mechanical stimulation on the structural and functional anisotropy of cardiomyocytes grown on a circular PDMS diaphragm.

Due to their immature morphology and functional immaturity, cardiomyocytes have limited use as an in vitro disease model of the native heart. Mechanical stimulation induces structural growth in cardiomyocytes in vitro by addressing the electrical-mechanical interactions between the tissues. However, current in vitro models are restricted in their capacity to replicate the milieu observed in natural myocardium. Herein, we proposed a Galinstan strain sensor integrated nanogrooved circular PDMS diaphragm to mimic the native cardiac tissues. The impact of combined topographical and mechanical stimulation on cultured cardiomyocytes at various strain areas on a circular PDMS diaphragm is studied in detail. An inverted microscope is used to image live cells and video acquisition to study the contractility of cultured cardiomyocytes. The structural changes of the cultured cardiomyocytes are investigated by its sarcomere length and connexin-43 (Cx43) expression using immunocytochemistry analysis. Cyclic strain is found to promote structural development in cultured cardiomyocytes, and diaphragms with nano-groove patterns displayed increased contractile activity and gene expression (sarcomere length ∼1.97 ± 0.03 µm and normalized Cx43-1.57) as compared to flat diaphragms (sarcomere length ∼1.82 ± 0.02 µm and normalized Cx43-1.32). The nanogrooved circular diaphragm exhibited distinct stretching mechanisms at various places, with the equibi-axial stretching regions providing the optimal structural growth and formation of natural myocardium at the diaphragm's center. Cardiomyocytes that are more mature have the potential to produce a more realistic in vitro cardiac model for disease modeling and medication development.

64 PI/PDMS hybrid cantilever arrays with an integrated strain sensor for a high-throughput drug toxicity screening application.

Herein, we propose a novel biosensing platform involving an array of 64 hybrid cantilevers and integrated strain sensors to measure the real-time contractility of the drug-treated cardiomyocytes (CMs). The strain sensor is integrated on the polyimide (PI) cantilever. To improve the strain sensor reliability and construct the engineered cardiac tissue, the nanogroove-patterned polydimethylsiloxane (PDMS) encapsulation layer is bonded on the PI cantilever. The preliminary sensing characteristics demonstrate the superior structural integrity, robustness, enhanced sensitivity, and repeatability of the proposed devices. The long-term durability and biocompatibility of the PI/PDMS hybrid cantilever is verified by evaluating the cell viability and contractility. We also validate the proposed biosensing platform for cardiotoxicity measurement by applying it to two specific cardiovascular drugs: quinidine and verapamil. In response to quinidine and verapamil, the engineered CMs exhibited negative inotropic and chronotropic effects. The fabricated cantilever device successfully detected the quinidine-induced adverse effects in CMs such as early after depolarization (EADs) and Torsade de points (TdP) in real-time. The array of hybrid cantilevers with integrated strain sensors has the potential to satisfy the need for innovative analytic platforms owing to its high throughput and simplified data analysis.

Mechanoadaptive organization of stress fiber subtypes in epithelial cells under cyclic stretches and stretch release.

Cyclic stretch applied to cells induces the reorganization of stress fibers. However, the correlation between the reorganization of stress fiber subtypes and strain-dependent responses of the cytoplasm and nucleus has remained unclear. Here, we investigated the dynamic involvement of stress fiber subtypes in the orientation and elongation of cyclically stretched epithelial cells. We applied uniaxial cyclic stretches at 5%, 10%, and 15% strains to cells followed by the release of the mechanical stretch. Dorsal, transverse arcs, and peripheral stress fibers were mainly involved in the cytoplasm responses whereas perinuclear cap fibers were associated with the reorientation and elongation of the nucleus. Dorsal stress fibers and transverse arcs rapidly responded within 15 min regardless of the strain magnitude to facilitate the subsequent changes in the orientation and elongation of the cytoplasm. The cyclic stretches induced the additional formation of perinuclear cap fibers and their increased number was almost maintained with a slight decline after 2-h-long stretch release. The slow formation and high stability of perinuclear cap fibers were linked to the slow reorientation kinetics and partial morphology recovery of nucleus in the presence or absence of cyclic stretches. The reorganization of stress fiber subtypes occurred in accordance with the reversible distribution of myosin II. These findings allowed us to propose a model for stretch-induced responses of the cytoplasm and nucleus in epithelial cells based on different mechanoadaptive properties of stress fiber subtypes.

Micro-patterned SU-8 cantilever integrated with metal electrode for enhanced electromechanical stimulation of cardiac cells.

Over the past few years, cardiac tissue engineering has undergone tremendous progress. Various in vitro methods have been developed to improve the accuracy in the result of drug-induced cardiac toxicity screening. Herein, we propose a novel SU-8 cantilever integrated with an electromechanical-stimulator to enhance the maturation of cultured cardiac cells. The simultaneous electromechanical stimulation significantly enhances the contraction force of the cardiomyocytes, thereby increasing cantilever displacement. Fluorescence microscopy analysis was performed to confirm the improved maturation of the cardiomyocytes. After the initial experiments, the contractile behaviors of the cultured cardiomyocytes were investigated by measuring the mechanical deformation of the SU-8 cantilever. Finally, the proposed electromechanical-stimulator-integrated SU-8 cantilever was used to evaluate the adverse effects of different cardiac vascular drugs, i.e., verapamil, lidocaine, and isoproterenol, on the cultured cardiomyocytes. The physiology of the cardiac-drug-treated cardiomyocytes was examined with and without electrical stimulation of the cardiomyocytes. The experimental results indicate that the proposed cantilever platform can be used as a predictive assay system for preliminary cardiac drug toxicity screening applications.

Development of a Next-Generation Biosensing Platform for Simultaneous Detection of Mechano- and Electrophysiology of the Drug-Induced Cardiomyocytes.

Detection of adverse effects of cardiac toxicity at an early stage by in vitro methods is crucial for the preclinical drug screening. Over the years, several kinds of biosensing platforms have been proposed by the scientific society for the detection of cardiac toxicity. However, the proposed tissue platforms have been optimized to measure either mechanophysiology or electrophysiology of the cardiomyocytes but not both. Herein, we demonstrate in detail our successful attempt toward developing a novel "multifunctional microphysiological system" also known as "organs-on-chips" to measure simultaneously the mechanical and electrical characteristics of cardiomyocytes in vitro. The proposed device can rapidly recognize drug-induced cardiovascular toxicity in real time, which is one of the most significant factors for drug discovery and postmarketing surveillance. We confirm that the proposed sensor delivers the direct relationship between the contraction force and cell impedance of cardiomyocytes under the influence of different cardiovascular drugs such as verapamil, astemizole, and lidocaine. The obtained assay results provide a great potential for a deep understanding of the drug effects on the cardiomyocytes in vitro.

Surface-patterned SU-8 cantilever arrays for preliminary screening of cardiac toxicity.

Arrays of a µgrooved SU-8 cantilever were utilized to analyze changes in the contraction force and beating frequency of cardiomyocytes in vitro. The longitudinally patterned µgrooves facilitates alignment of cardiomyocytes on top of the SU-8 cantilever, which increases the contraction force of cardiomyocytes by a factor of about 2.5. The bending displacement of the SU-8 cantilever was precisely measured in nanoscale using a laser-based measurement system combined with a motorized xyz stage. The cantilever displacement due to contraction of the cardiomyocytes showed the maximum on day 8 after their cultivation. Following preliminary experiments, Isoproterenol, Verapamil, and Astemizole were used to investigate the effect of drug toxicity on the physiology of cardiomyocytes. The experimental results indicated that 1 µM of Isoproterenol treatment increased contraction force and beating frequencies of cardiomyocytes by 30% and 200%, respectively, whereas 500 nM of Verapamil treatment decreased contraction force and beating frequencies of cardiomyocytes by 56% and 42%, respectively. A concentration of less than 5 nM of the hERG channel suppression drug Astemizole did not change the contraction forces in the displacement but slightly decreased the beating frequencies. However, irregular or abnormal heartbeats were observed at Astemizole concentrations of 5 nM and higher. We experimentally conformed that the proposed SU-8 cantilever arrays combined with the laser-based measurement systems has the great potential for a high-throughput drug toxicity screening system in future.

Graphene/polydimethylsiloxane nanocomposite strain sensor.

The objective of this research is to fabricate graphene nanopowder composites based on polydimethylsiloxane (PDMS) and to characterize the gauge factor of the graphene/PDMS composites for the use of strain sensors. The fabrication of graphene/PDMS composites can be accomplished by simple sonication and micro molding processes. We found that the measured gauge factors strongly depend on the concentration of graphene flakes in the composites. Obtained gauge factor of the graphene/PDMS composite strain sensor reached about 233 at a graphene concentration of 8.33 vol.%, which was measured within a strain range of 2%.

Effect of replicated polymeric substrate with lotus surface structure on adipose-derived stem cell behaviors.

We fabricated polystyrene substrates with lotus leaf surface structure (LLSS) and investigated cell behaviors, including attachment, morphology, proliferation, and differentiation of adipose-derived stem cells (ASCs) on them. Compared to the flat substrate, the LLSS substrate induced higher cell attachment rate, but did not significantly change the cell proliferation rate. In addition, ASCs on the LLSS substrate exhibited relatively narrower spreading morphology and less organized cytoskeleton, there by resulting in smaller sizes of cells than those on the flat substrate. According to histochemical staining and RT-PCR analysis, the LLSS substrate induced higher adipogenic differentiation of ASCs than the flat substrate, while chondrogenic and osteogenic differentiation were decreased.