Advanced Materials and Sensors for Microphysiological Systems: Focus on Electronic and Electrooptical Interfaces.

Advanced in vitro cell culture systems or microphysiological systems (MPSs), including microfluidic organ-on-a-chip (OoC), are breakthrough technologies in biomedicine. These systems recapitulate features of human tissues outside of the body. They are increasingly being used to study the functionality of different organs for applications such as drug evolutions, disease modeling, and precision medicine. Currently, developers and endpoint users of these in vitro models promote how they can replace animal models or even be a better ethically neutral and humanized alternative to study pathology, physiology, and pharmacology. Although reported models show a remarkable physiological structure and function compared to the conventional 2D cell culture, they are almost exclusively based on standard passive polymers or glass with none or minimal real-time stimuli and readout capacity. The next technology leap in reproducing in vivo-like functionality and real-time monitoring of tissue function could be realized with advanced functional materials and devices. This review describes the currently reported electronic and optical advanced materials for sensing and stimulation of MPS models. In addition, an overview of multi-sensing for Body-on-Chip platforms is given. Finally, one gives the perspective on how advanced functional materials could be integrated into in vitro systems to precisely mimic human physiology.

Dual effect of TAT functionalized DHAH lipid nanoparticles with neurotrophic factors in human BBB and microglia cultures.

BACKGROUND: Neurodegenerative diseases (NDs) are an accelerating global health problem. Nevertheless, the stronghold of the brain- the blood-brain barrier (BBB) prevents drug penetrance and dwindles effective treatments. Therefore, it is crucial to identify Trojan horse-like drug carriers that can effectively cross the blood-brain barrier and reach the brain tissue. We have previously developed polyunsaturated fatty acids (PUFA)-based nanostructured lipid carriers (NLC), namely DHAH-NLC. These carriers are modulated with BBB-permeating compounds such as chitosan (CS) and trans-activating transcriptional activator (TAT) from HIV-1 that can entrap neurotrophic factors (NTF) serving as nanocarriers for NDs treatment. Moreover, microglia are suggested as a key causative factor of the undergoing neuroinflammation of NDs. In this work, we used in vitro models to investigate whether DHAH-NLCs can enter the brain via the BBB and investigate the therapeutic effect of NTF-containing DHAH-NLC and DHAH-NLC itself on lipopolysaccharide-challenged microglia. METHODS: We employed human induced pluripotent stem cell-derived brain microvascular endothelial cells (BMECs) to capitalize on the in vivo-like TEER of this BBB model and quantitatively assessed the permeability of DHAH-NLCs. We also used the HMC3 microglia cell line to assess the therapeutic effect of NTF-containing DHAH-NLC upon LPS challenge. RESULTS: TAT-functionalized DHAH-NLCs successfully crossed the in vitro BBB model, which exhibited high transendothelial electrical resistance (TEER) values (≈3000 Ω*cm2). Specifically, the TAT-functionalized DHAH-NLCs showed a permeability of up to 0.4% of the dose. Furthermore, using human microglia (HMC3), we demonstrate that DHAH-NLCs successfully counteracted the inflammatory response in our cultures after LPS challenge. Moreover, the encapsulation of glial cell-derived neurotrophic factor (GNDF)-containing DHAH-NLCs (DHAH-NLC-GNDF) activated the Nrf2/HO-1 pathway, suggesting the triggering of the endogenous anti-oxidative system present in microglia. CONCLUSIONS: Overall, this work shows that the TAT-functionalized DHAH-NLCs can cross the BBB, modulate immune responses, and serve as cargo carriers for growth factors; thus, constituting an attractive and promising novel drug delivery approach for the transport of therapeutics through the BBB into the brain.

Electrochemical control of growth factor presentation to steer neural stem cell differentiation.

Let it grow: The conjugated polymer poly(3,4-ethylenedioxythiophene) (PEDOT) was synthesized with heparin as the counterion to form a cell culture substrate. The surface of PEDOT:heparin in the neutral state associated biologically active growth factors. Electrochemical in situ oxidation of PEDOT during live cell culture decreased the bioavailability of the growth factor and created an exact onset of neural stem cell differentiation.

Sorption of Neuropsychopharmaca in Microfluidic Materials for In Vitro Studies.

Sorption (i.e., adsorption and absorption) of small-molecule compounds to polydimethylsiloxane (PDMS) is a widely acknowledged phenomenon. However, studies to date have largely been conducted under atypical conditions for microfluidic applications (lack of perfusion, lack of biological fluids, etc.), especially considering biological studies such as organs-on-chips where small-molecule sorption poses the largest concern. Here, we present an in-depth study of small-molecule sorption under relevant conditions for microphysiological systems, focusing on a standard geometry for biological barrier studies that find application in pharmacokinetics. We specifically assess the sorption of a broad compound panel including 15 neuropsychopharmaca at in vivo concentration levels. We consider devices constructed from PDMS as well as two material alternatives (off-stoichiometry thiol-ene-epoxy, or tape/polycarbonate laminates). Moreover, we study the much neglected impact of peristaltic pump tubing, an essential component of the recirculating systems required to achieve in vivo-like perfusion shear stresses. We find that the choice of the device material does not have a significant impact on the sorption behavior in our barrier-on-chip-type system. Our PDMS observations in particular suggest that excessive compound sorption observed in prior studies is not sufficiently described by compound hydrophobicity or other suggested predictors. Critically, we show that sorption by peristaltic tubing, including the commonly utilized PharMed BPT, dominates over device sorption even on an area-normalized basis, let alone at the typically much larger tubing surface areas. Our findings highlight the importance of validating compound dosages in organ-on-chip studies, as well as the need for considering tubing materials with equal or higher care than device materials.

Bactericidal surfaces prepared by femtosecond laser patterning and layer-by-layer polyelectrolyte coating.

Antimicrobial surfaces are important in medical, clinical, and industrial applications, where bacterial infection and biofouling may constitute a serious threat to human health. Conventional approaches against bacteria involve coating the surface with antibiotics, cytotoxic polymers, or metal particles. However, these types of functionalization have a limited lifetime and pose concerns in terms of leaching and degradation of the coating. Thus, there is a great interest in developing long-lasting and non-leaching bactericidal surfaces. To obtain a bactericidal surface, we combine micro and nanoscale patterning of borosilicate glass surfaces by ultrashort pulsed laser irradiation and a non-leaching layer-by-layer polyelectrolyte modification of the surface. The combination of surface structure and surface charge results in an enhanced bactericidal effect against both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria. The laser patterning and the layer-by-layer modification are environmentally friendly processes that are applicable to a wide variety of materials, which makes this method uniquely suited for fundamental studies of bacteria-surface interactions and paves the way for its applications in a variety of fields, such as in hygiene products and medical devices.

Conjugated Polymers for Assessing and Controlling Biological Functions.

The field of organic bioelectronics is advancing rapidly in the development of materials and devices to precisely monitor and control biological signals. Electronics and biology can interact on multiple levels: organs, complex tissues, cells, cell membranes, proteins, and even small molecules. Compared to traditional electronic materials such as metals and inorganic semiconductors, conjugated polymers (CPs) have several key advantages for biological interactions: tunable physiochemical properties, adjustable form factors, and mixed conductivity (ionic and electronic). Herein, the use of CPs in five biologically oriented research topics, electrophysiology, tissue engineering, drug release, biosensing, and molecular bioelectronics, is discussed. In electrophysiology, implantable devices with CP coating or CP-only electrodes are showing improvements in signal performance and tissue interfaces. CP-based scaffolds supply highly favorable static or even dynamic interfaces for tissue engineering. CPs also enable delivery of drugs through a variety of mechanisms and form factors. For biosensing, CPs offer new possibilities to incorporate biological sensing elements in a conducting matrix. Molecular bioelectronics is today used to incorporate (opto)electronic functions in living tissue. Under each topic, the limits of the utility of CPs are discussed and, overall, the major challenges toward implementation of CPs and their devices to real-world applications are highlighted.

Alignment of a conjugated polymer onto amyloid-like protein fibrils.

The amyloid-like fibril is a biomolecular nanowire template of very high stability. Here we describe the coordination of a conjugated polyelectrolyte, poly(thiophene acetic acid) (PTAA), to bovine insulin fibrils with widths of <10 nm and lengths of up to more than 10 microm. Fibrils complexed with PTAA are aligned on surfaces through molecular combing and transfer printing. Single-molecule spectroscopy techniques are applied to chart spectral variation in the emission of these wires. When these results are combined with analysis of the polarization of the emitted light, we can conclude that the polymer chains are preferentially aligned along the fibrillar axis.

Control of neural stem cell survival by electroactive polymer substrates.

Stem cell function is regulated by intrinsic as well as microenvironmental factors, including chemical and mechanical signals. Conducting polymer-based cell culture substrates provide a powerful tool to control both chemical and physical stimuli sensed by stem cells. Here we show that polypyrrole (PPy), a commonly used conducting polymer, can be tailored to modulate survival and maintenance of rat fetal neural stem cells (NSCs). NSCs cultured on PPy substrates containing different counter ions, dodecylbenzenesulfonate (DBS), tosylate (TsO), perchlorate (ClO(4)) and chloride (Cl), showed a distinct correlation between PPy counter ion and cell viability. Specifically, NSC viability was high on PPy(DBS) but low on PPy containing TsO, ClO(4) and Cl. On PPy(DBS), NSC proliferation and differentiation was comparable to standard NSC culture on tissue culture polystyrene. Electrical reduction of PPy(DBS) created a switch for neural stem cell viability, with widespread cell death upon polymer reduction. Coating the PPy(DBS) films with a gel layer composed of a basement membrane matrix efficiently prevented loss of cell viability upon polymer reduction. Here we have defined conditions for the biocompatibility of PPy substrates with NSC culture, critical for the development of devices based on conducting polymers interfacing with NSCs.

Imaging of the 3D nanostructure of a polymer solar cell by electron tomography.

Electron tomography has been used for analyzing the active layer in a polymer solar cell, a bulk heterojunction of an alternating copolymer of fluorene and a derivative of fullerene. The method supplies a three-dimensional representation of the morphology of the film, where domains with different scattering properties may be distinguished. The reconstruction shows good contrast between the two phases included in the film and demonstrates that electron tomography is an adequate tool for investigations of the three-dimensional nanostructure of the amorphous materials used in polymer solar cells.

Electrochemical devices made from conducting nanowire networks self-assembled from amyloid fibrils and alkoxysulfonate PEDOT.

Proteins offer an almost infinite number of functions and geometries for building nanostructures. Here we have focused on amyloid fibrillar proteins as a nanowire template and shown that these fibrils can be coated with the highly conducting polymer alkoxysulfonate PEDOT through molecular self-assembly in water. Transmission electron microscopy and atomic force microscopy show that the coated fibers have a diameter around 15 nm and a length/thickness aspect ratio >1:1000 . We have further shown that networks of the conducting nanowires are electrically and electrochemically active by constructing fully functional electrochemical transistors with nanowire networks, operating at low voltages between 0 and 0.5 V.

Enhanced current efficiency from bio-organic light-emitting diodes using decorated amyloid fibrils with conjugated polymer.

We demonstrate the use of self-assembled bionanostructures in polymer light-emitting diodes. Amyloid fibrils formed by protein misfolding were decorated with a soluble luminescent conjugated polymer. This conjugated polymer complex with amyloid fibrils was used as the active layer in a light emitting diode, resulting in a 10-fold increase in external quantum efficiency compared with pristine polymer, because of improved carrier injection.

Studies of luminescent conjugated polythiophene derivatives: enhanced spectral discrimination of protein conformational states.

Improved probes for amyloid fibril formation are advantageous for the early detection and better understanding of this disease-associated process. Here, we report a comparative study of eight luminescent conjugated polythiophene derivates (LCPs) and their discrimination of a protein (insulin) in the native or amyloid-like fibrillar state. For two of the LCPs, the synthesis is reported. Compared to their monomer-based analogues, trimer-based LCPs showed significantly better optical signal specificity for amyloid-like fibrils, seen from increased quantum yield and spectral shift. The trimer-based LCPs alone were highly quenched and showed little interaction with native insulin, as seen from analytical ultracentrifugation and insignificant spectral differences from the trimer-based LCP in buffered and native protein solution. Hence, the trimer-based LCPs showed enhanced discrimination between the amyloid-like fibrillar state and the corresponding native protein.

Single molecular imaging and spectroscopy of conjugated polyelectrolytes decorated on stretched aligned DNA.

DNA is the prototype template for building nanoelectronic devices by self-assembly. The electronic functions are made possible by coordinating electronic polymer chains to DNA. This paper demonstrates two methods for fabrication of aligned and ordered DNA nanowires complexed with conjugated polyelectrolytes (CPEs). The complex can be formed either in solution prior to stretching or after stretching of the bare DNA on a surface. Molecular combing was used to stretch the complexes on surface energy patterned surfaces, and PMMA for the bare DNA. Single molecular spectroscopy, in fluorescence, and microscopy, in atomic force microscopy, give evidence for coordination of the short CPE chains to the aligned DNA.

Conjugated polyelectrolytes: conformation-sensitive optical probes for detection of amyloid fibril formation.

The in vivo deposition of amyloid fibrils is a hallmark of many devastating diseases known as the amyloidoses. Amyloid formation in vitro may also complicate production of proteins in the biotechnology industry. Simple, sensitive, and versatile tools that detect the fibrillar conformation of amyloidogenic proteins are thus of great importance. We have developed a negatively charged conjugated polyelectrolyte that displays different characteristic optical changes, detected visually or by absorption and emission, depending on whether the protein with which it forms a complex is in its native state or amyloid fibril conformation. This simple, rapid, and novel methodology was applied here to two amyloidogenic proteins, insulin and lysozyme, and its validity for detection of their fibrillar conformation was verified by currently used methods such as circular dichroism, transmission electron microscopy, and Congo red absorption.

Synthesis of a regioregular zwitterionic conjugated oligoelectrolyte, usable as an optical probe for detection of amyloid fibril formation at acidic pH.

Changes of the optical properties of conjugated polyelectrolytes have been utilized to monitor noncovalent interactions between biomolecules and the conjugated polyelectrolytes in sensor applications. A regioregular, zwitterionic conjugated oligoelectrolyte was synthesized in order to create a probe with a defined set of optical properties and hereby facilitate interpretation of biomolecule-oligoelectrolyte interactions. The synthesized oligoelectrolyte was used at acidic pH as a novel optical probe to detect amyloid fibril formation of bovine insulin and chicken lysozyme. Interaction of the probe with formed amyloid fibrils results in changes of the geometry and the electronic structure of the oligoelectrolyte chains, which were monitored with absorption and emission spectroscopy.