ION Thallos-HTL: a fluorescent thallium indicator that enables cell-selective and localizable thallium flux assays.

Ion channels are essential proteins for all organisms. Electrophysiology is a useful and commonly employed method to study ion channels, however there is a need for operationally simpler, cost-effective and higher throughput techniques to study ion channel functions in their native environments. Fluorescent ion indicators, such as Fluo-4 and Thallos, have been used for decades to study ion channel activity by measuring the flux of ions through channels of interest. In this work, we present ION Thallos-HTL, a thallium indicator that can be localized using HaloTag technology. This novel indicator enables specific labeling of cells and intracellular compartments in live cells and responds to changes in thallium concentration within these environments. We demonstrate the utility of ION Thallos-HTL by conducting a thallium flux assay using high-throughput instrumentation in a mixed cell population where some cells are expressing HaloTag and some are not.

Laser-imprinting of micro-3D printed protein hydrogels enables real-time independent modification of substrate topography and elastic modulus.

Independent control over the Young's modulus and topography of a hydrogel cell culture substrate is necessary to characterize how attributes of its adherent surface affect cellular responses. Arbitrary, real-time manipulation of these parameters at the micron scale would further provide cellular biologists and bioengineers with the tools to study and control numerous highly dynamic behaviors including cellular adhesion, motility, metastasis, and differentiation. Although physical, chemical, thermal, and light-based strategies have been developed to influence Young's modulus and topography of hydrogel substrates, independent control of these physical attributes has remained elusive, spatial resolution is often limited, and features commonly must be pre-patterned. We recently reported a strategy in which biomaterials having specified three-dimensional (3D) morphologies are micro-3D printed in a two-step process: laser-scanning bioprinting of a protein-based hydrogel, followed by biocompatible hydrogel re-scanning to create microscale imprinted features at user-defined times. In this approach, a pulsed near-infrared laser beam is focused within the printed hydrogel to promote matrix contraction through multiphoton crosslinking, where scanning the laser focus projects a user-defined topographical pattern on the surface without subjecting the hydrogel-solution interface to damaging light intensities. Here, we extend this strategy, demonstrating the ability to decouple dynamic topographical changes from changes in hydrogel Young's modulus at the substrate surface by increasing the isolation distance between the surface and re-scanning planes. Using atomic force microscopy, we show that robust topographic changes can be imposed without altering the Young's modulus measured at the substrate surface by scanning at a depth of greater than ~6 µm. Transmission electron microscopy of hydrogel thin sections reveals changes to hydrogel porosity and density distribution within scanned regions, and that such changes to the hydrogel matrix are highly localized to regions of laser exposure. These results represent valuable new capabilities for deconvolving the effects of substrate dynamic physical attributes on the behavior of adherent cells.

Assessing the range of enzymatic and oxidative tunability for biosensor design.

Development of multi-functional materials and biosensors that can achieve an in situ response designed by the user is a current need in the biomaterials field, especially in complex biological environments, such as inflammation, where multiple enzymatic and oxidative signals are present. In the past decade, there has been extensive research and development of materials chemistries for detecting and monitoring enzymatic activity, as well as for releasing therapeutic and diagnostic agents in regions undergoing oxidative stress. However, there has been limited development of materials in the context of enzymatic and oxidative triggers together, despite their closely tied and overlapping mechanisms. With research focusing on enzymatically and oxidatively triggered materials separately, these systems may be inadequate in monitoring the complexity of inflammatory environments, thus limiting in vivo translatability and diagnostic accuracy. The intention of this review is to highlight a variety of enzymatically and oxidatively triggered materials chemistries to draw attention to the range of synthetic tunability available for the construction of novel biosensors with a spectrum of programmed responses. We focus our discussion on several types of macromolecular sensors, generally classified by the causative material response driving ultimate signal detection. This includes sensing based on degradative processes, conformational changes, supramolecular assembly/disassembly, and nanomaterial interactions, among others. We see each of these classes providing valuable tools toward coalescing current gaps in the biosensing field regarding specificity, selectivity, sensitivity, and flexibility in application. Additionally, by considering the materials chemistry of enzymatically and oxidatively triggered biomaterials in tandem, we hope to encourage synthesis of new biosensors that capitalize on their synergistic roles and overlapping mechanisms in inflammatory environments for applications in disease diagnosis and monitoring.

2-Amino-3'-dialkylaminobiphenyl-based fluorescent intracellular probes for nitric oxide surrogate N2O3.

Fluorescent probes for nitric oxide (NO), or more frequently for its oxidized surrogate dinitrogen trioxide (N2O3), have enabled scientists to study the contributions of this signaling molecule to many physiological processes. Seeking to improve upon limitations of other probes, we have developed a family of fluorescent probes based on a 2-amino-3'-dialkylaminobiphenyl core. This core condenses with N2O3 to form benzo[c]cinnoline structures, incorporating the analyte into the newly formed fluorophore, which results in product fluorescence with virtually no background contribution from the initial probe. We varied the substituents in the core in order to optimize both the reactivity of the probes with N2O3 and their cinnoline products' fluorescence wavelengths and brightness. The top candidates were then applied to cultured cells to verify that they could respond to NO within cellular milieus, and the top performer, NO530, was compared with a "gold standard" commercial probe, DAF-FM, in a macrophage-derived cell line, RAW 264.7, stimulated to produce NO. NO530 demonstrated similar or better sensitivity and higher selectivity for NO than DAF, making it an attractive potential alternative for NO tracking in various applications.

Poly-d-lysine coated nanoparticles to identify pro-inflammatory macrophages.

Identifying pro-inflammatory macrophages (M1) is of immense importance to diagnose, monitor, and treat various pathologies. In addition, adoptive cell therapies, where harvested cells are isolated, modified to express an M1-like phenotype, then re-implanted to the patient, are also becoming more prevalent to treat diseases such as cancer. In a step toward identifying, labeling, and monitoring macrophage phenotype for adoptive cell therapies, we developed a reactive oxygen species (ROS)-sensitive, gold nanoparticle (AuNP) that fluorescently labels M1 macrophages. AuNPs are electrostatically coated with a proteolysis resistant, fluorescein isothiocyanate-conjugated, poly-d-lysine (PDL-FITC) that is susceptible to backbone cleavage by ROS. When PDL-FITC is bound to AuNPs, fluorescence is quenched via a combination of nanoparticle surface (NSET) and Forster resonance (FRET) energy transfer mechanisms. Upon ROS-induced cleavage of PDL-FITC, up to a 7-fold change in fluorescence is demonstrated. PDL-FITC AuNPs were loaded into RAW 264.7 macrophages (RAWs) and primary bone marrow- derived macrophages (BMDMs) prior to in vitro polarization. For both cell types, detectable differences in intracellular fluorescence were observed between M1 polarized and non-stimulated (M0) control groups after 24 h using both confocal imaging and flow cytometry. PDL-FITC AuNPs can potentially be useful in identifying M1 macrophages within diverse cell populations and provide longitudinal macrophage response data to external cues.

In Vivo Photoacoustic Tracking of Mesenchymal Stem Cell Viability.

Adult stem cell therapy has demonstrated improved outcomes for treating cardiovascular diseases in preclinical trials. The development of imaging tools may increase our understanding of the mechanisms of stem cell therapy, and a variety of imaging tools have been developed to image transplanted stem cells in vivo; however, they lack the ability to interrogate stem cell function longitudinally. Here, we report the use of a nanoparticle-based contrast agent that can track stem cell viability using photoacoustic imaging. The contrast agent consists of inert gold nanorods coated with IR775c, a reactive oxygen species (ROS) sensitive near-infrared dye. Upon cell death, stem cells produce ROS to degrade the cell. Using this feature of stem cells, the viability can be measured by comparing the IR775c signal to the ROS insensitive gold nanorod signal, which can also be used to track stem cell location. The nanoprobe was successfully loaded into mesenchymal stem cells (MSCs), and then, MSCs were transplanted into the lower limb of a mouse and imaged using combined ultrasound and photoacoustic imaging. MSC viability was assessed using the nanoprobe and displayed significant cell death within 24 h and an estimated 5% viability after 10 days. This nanoparticle system allows for longitudinal tracking of MSC viability in vivo with high spatial and temporal resolution which other imaging modalities currently cannot achieve.

In Situ Imprinting of Topographic Landscapes at the Cell-Substrate Interface.

In their native environments, adherent cells encounter dynamic topographical cues involved in promoting differentiation, orientation, and migration. Ideally, such processes would be amenable to study in cell culture using tools capable of imposing dynamic, arbitrary, and reversible topographic features without perturbing environmental conditions or causing chemical and/or structural disruptions to the substrate surface. To address this need, we report here development of an in vitro strategy for challenging cells with dynamic topographical experiences in which protein-based hydrogel substrate surfaces are modified in real time by positioning a pulsed, near-infrared laser focus within the hydrogel, promoting chemical cross-linking which results in local contraction of the protein matrix. Scanning the laser focus through arbitrary patterns directed by a dynamic reflective mask creates an internal contraction pattern that is projected onto the hydrogel surface as features such as rings, pegs, and grooves. By subjecting substrates to a sequence of scan patterns, we show that topographic features can be created, then eliminated or even reversed. Because laser-induced shrinkage can be confined to 3D voxels isolated from the cell-substrate interface, hydrogel modifications are made without damaging cells or disrupting the chemical or structural integrity of the surface.

Multiphoton microfabrication of conducting polymer-based biomaterials.

We report the application of multiphoton microfabrication to prepare conducting polymer (CP)-based biomaterials that were capable of drug delivery and interacting with brain tissue ex vivo, thereby highlighting the potential of multiphoton lithography to prepare electroactive biomaterials which may function as implantable neural biointerfaces (e.g. electrodes).

Regioselective Localization and Tracking of Biomolecules on Single Gold Nanoparticles.

Selective localization of biomolecules at the hot spots of a plasmonic nanoparticle is an attractive strategy to exploit the light-matter interaction due to the high field concentration. Current approaches for hot spot targeting are time-consuming and involve prior knowledge of the hot spots. Multiphoton plasmonic lithography is employed to rapidly immobilize bovine serum albumin (BSA) hydrogel at the hot spot tips of a single gold nanotriangle (AuNT). Regioselectivity and quantity control by manipulating the polarization and intensity of the incident laser are also established. Single AuNTs are tracked using dark-field scattering spectroscopy and scanning electron microscopy to characterize the regioselective process. Fluorescence lifetime measurements further confirm BSA immobilization on the AuNTs. Here, the AuNT-BSA hydrogel complexes, in conjunction with single-particle optical monitoring, can act as a framework for understanding light-molecule interactions at the subnanoparticle level and has potential applications in biophotonics, nanomedicine, and life sciences.

Conducting polymer-based multilayer films for instructive biomaterial coatings.

AIM: To demonstrate the design, fabrication and testing of conformable conducting biomaterials that encourage cell alignment. MATERIALS & METHODS: Thin conducting composite biomaterials based on multilayer films of poly(3.4-ethylenedioxythiophene) derivatives, chitosan and gelatin were prepared in a layer-by-layer fashion. Fibroblasts were observed with fluorescence microscopy and their alignment (relative to the dipping direction and direction of electrical current passed through the films) was determined using ImageJ. RESULTS: Fibroblasts adhered to and proliferated on the films. Fibroblasts aligned with the dipping direction used during film preparation and this was enhanced by a DC current. CONCLUSION: We report the preparation of conducting polymer-based films that enhance the alignment of fibroblasts on their surface which is an important feature of a variety of tissues.