Development of an Optical Nanosensor Incorporating a pH-Sensitive Quencher Dye for Potassium Imaging.

One of the key challenges in the design of a sensor for measuring extracellular changes in potassium concentration is selectivity against the competing cation, sodium. Here, we present an optode-based nanosensor selective to potassium ions, owing to the addition of a pH-sensitive quencher molecule paired with a static fluorophore. The nanosensor was fabricated using emulsification and characterized in solution by absorbance and fluorescence spectroscopy. The resulting nanosensor detected potassium with nearly 1 order of magnitude higher selectivity compared to our chromoionophore-based optode nanosensors. In addition to the improved selectivity, the nanosensor has the following properties required for measurements in a biological environment: (1) a physiologically relevant dynamic range, (2) response to potassium ions at a physiological ionic strength, and (3) response to serum potassium in the presence of fouling biological components. The potassium nanosensor described in this study is envisioned to have application in cellular imaging and drug screening.

Implantable nanosensors: toward continuous physiologic monitoring.

Continuous physiologic monitoring would add greatly to both home and clinical medical treatment for chronic conditions. Implantable nanosensors are a promising platform for designing continuous monitoring systems. This Feature reviews design considerations and current approaches toward such devices.

Antimicrobial effects of nanofiber poly(caprolactone) tissue scaffolds releasing rifampicin.

This study quantified the antibiotic release kinetics and subsequent bactericidal efficacy of rifampicin (RIF) against Gram-positive and Gram-negative bacteria under in vitro static conditions. Antibiotic-loaded scaffolds were fabricated by electrospinning poly(caprolactone) (PCL) with 10% or 20% (w/w) RIF. Scaffold fiber diameter and RIF loading were characterized, and RIF release kinetics were measured. RIF-releasing and RIF-free scaffolds were inoculated with Pseudomonas aeruginosa and Staphylococcus epidermidis, and the suspended concentration live and dead bacteria were determined by fluorescent microscopy. Adherent bacteria and biofilm formation were examined using scanning electron microscopy. Mean fiber diameters were 557 ± 399 nm for RIF-free, 402 ± 225 nm for 10% RIF, and 665 ± 402 nm for 20% RIF scaffolds. RIF release kinetics exhibited a short-burst release during the first hour, followed by a 7 h, zero-order release during which both RIF scaffolds released ~50% of their initial RIF mass loading. P. aeruginosa and S. epidermidis suspended cell populations proliferated in accordance with logarithmic growth models when exposed to control scaffolds; however both RIF-containing scaffolds completely inhibited bacterial growth in suspension and, subsequently, prevented biofilm formation within the scaffolds through the first 6 h.

Ion-Switchable Quantum Dot Förster Resonance Energy Transfer Rates in Ratiometric Potassium Sensors.

The tools for optically imaging cellular potassium concentrations in real-time are currently limited to a small set of molecular indicator dyes. Quantum dot-based nanosensors are more photostable and tunable than organic indicators, but previous designs have fallen short in size, sensitivity, and selectivity. Here, we introduce a small, sensitive, and selective nanosensor for potassium measurements. A dynamic quencher modulates the fluorescence emitted by two different quantum dot species to produce a ratiometric signal. We characterized the potassium-modulated sensor properties and investigated the photonic interactions within the sensors. The quencher's protonation changes in response to potassium, which modulates its Förster radiative energy transfer rate and the corresponding interaction radii with each quantum dot species. The nanosensors respond to changes in potassium concentrations typical of the cellular environment and thus provide a promising tool for imaging potassium fluxes during biological events.

Enzyme-linked DNA dendrimer nanosensors for acetylcholine.

It is currently difficult to measure small dynamics of molecules in the brain with high spatial and temporal resolution while connecting them to the bigger picture of brain function. A step towards understanding the underlying neural networks of the brain is the ability to sense discrete changes of acetylcholine within a synapse. Here we show an efficient method for generating acetylcholine-detecting nanosensors based on DNA dendrimer scaffolds that incorporate butyrylcholinesterase and fluorescein in a nanoscale arrangement. These nanosensors are selective for acetylcholine and reversibly respond to levels of acetylcholine in the neurophysiological range. This DNA dendrimer architecture has the potential to overcome current obstacles to sensing in the synaptic environment, including the nanoscale size constraints of the synapse and the ability to quantify the spatio-temporal fluctuations of neurotransmitter release. By combining the control of nanosensor architecture with the strategic placement of fluorescent reporters and enzymes, this novel nanosensor platform can facilitate the development of new selective imaging tools for neuroscience.

Polymer-free optode nanosensors for dynamic, reversible, and ratiometric sodium imaging in the physiological range.

This work introduces a polymer-free optode nanosensor for ratiometric sodium imaging. Transmembrane ion dynamics are often captured by electrophysiology and calcium imaging, but sodium dyes suffer from short excitation wavelengths and poor selectivity. Optodes, optical sensors composed of a polymer matrix with embedded sensing chemistry, have been translated into nanosensors that selectively image ion concentrations. Polymer-free nanosensors were fabricated by emulsification and were stable by diameter and sensitivity for at least one week. Ratiometric fluorescent measurements demonstrated that the nanosensors are selective for sodium over potassium by ~1.4 orders of magnitude, have a dynamic range centered at 20 mM, and are fully reversible. The ratiometric signal changes by 70% between 10 and 100 mM sodium, showing that they are sensitive to changes in sodium concentration. These nanosensors will provide a new tool for sensitive and quantitative ion imaging.

Mineralization content alters osteogenic responses of bone marrow stromal cells on hydroxyapatite/polycaprolactone composite nanofiber scaffolds.

Synthetic tissue scaffolds have a high potential impact for patients experiencing osteogenesis imperfecta. Using electrospinning, tissue scaffolds composed of hydroxyapatite/polycaprolactone (HAp/PCL) composite nanofibers were fabricated with two different HAp concentrations-1% and 10% of the solid scaffold weight. After physico-chemical scaffold characterization, rat bone marrow stromal cells were cultured on the composite scaffolds in maintenance medium and then in osteogenic medium. Quantitative PCR, colorimetric assays, immunofluorescent labeling, and electron microscopy measured osteogenic cell responses to the HAp/PCL scaffolds. In maintenance conditions, both Hap/PCL scaffolds and control scaffolds supported cell colonization through seven days with minor differences. In osteogenic conditions, the 10% HAp scaffolds exhibited significantly increased ALP assay levels at week 3, consistent with previous reports. However, qPCR analysis demonstrated an overall decrease in bone matrix-associated genes on Hap/PCL scaffolds. Osteopontin and osteocalcin immunofluorescent microscopy revealed a trend that both mineralized scaffolds had greater amounts of both proteins, though qPCR results indicated the opposite trend for osteopontin. Additionally, type I collagen expression decreased on HAp scaffolds. These results indicate that cells are sensitive to minor changes in mineral content within nanofibers, even at just 1% w/w, and elucidating the sensing mechanism may lead to optimized osteogenic scaffold designs.

Dynamic mechanical analysis and biomineralization of hyaluronan-polyethylene copolymers for potential use in osteochondral defect repair.

Treatment options for damaged articular cartilage are limited due to its lack of vasculature and its unique viscoelastic properties. This study was the first to fabricate a hyaluronan (HA)-polyethylene copolymer for potential use in the replacement of articular cartilage and repair of osteochondral defects. Amphiphilic graft copolymers consisting of HA and high-density polyethylene (HA-co-HDPE) were fabricated with 10, 28 and 50 wt.% HA. Dynamic mechanical analysis was used to assess the effect of varying constituent weight ratios on the viscoelastic properties of HA-co-HDPE materials. The storage moduli of HA-co-HDPE copolymers ranged from 2.4 to 15.0 MPa at physiological loading frequencies. The viscoelastic properties of the HA-co-HDPE materials were significantly affected by varying the wt.% of HA and/or crosslinking of the HA constituent. Cytotoxicity and the ability of the materials to support mineralization were evaluated in the presence of bone marrow stromal cells. HA-co-HDPE materials were non-cytotoxic, and calcium and phosphorus were present on the surface of the HA-co-HDPE materials 2 weeks after osteogenic differentiation of the bone marrow stromal cells. This study is the first to measure the viscoelastic properties and osseocompatibility of HA-co-HDPE for potential use in orthopedic applications.

Osteogenic differentiation of bone marrow stromal cells on poly(epsilon-caprolactone) nanofiber scaffolds.

Nanofiber poly(epsilon-caprolactone) (PCL) scaffolds were fabricated by electrospinning, and their ability to enhance the osteoblastic behavior of marrow stromal cells (MSCs) in osteogenic media was investigated. MSCs were isolated from Wistar rats and cultured on nanofiber scaffolds to assess short-term cytocompatibility and long-term phenotypic behavior. Smooth PCL substrates were used as control surfaces. The short-term cytocompatibility results indicated that nanofiber scaffolds supported greater cell adhesion and viability compared with control surfaces. In osteogenic conditions, MSCs cultured on nanofiber scaffolds also displayed increased levels of alkaline phosphatase activity for 3 weeks of culture. Calcium phosphate mineralization was substantially accelerated on nanofiber scaffolds compared to control surfaces as indicated through von Kossa and calcium staining, scanning electron microscopy and energy-dispersive X-ray spectroscopy. Increased levels of intra- and extracellular levels of osteocalcin and osteopontin were observed on nanofiber scaffolds using immunofluorescence techniques after 3 weeks of culture. These results demonstrate the enhanced tissue regeneration property of nanofiber scaffolds, which may be of potential use for engineering osteogenic scaffolds for orthopedic applications.

Bone tissue engineering: a review in bone biomimetics and drug delivery strategies.

Critical-sized defects in bone, whether induced by primary tumor resection, trauma, or selective surgery have in many cases presented insurmountable challenges to the current gold standard treatment for bone repair. The primary purpose of a tissue-engineered scaffold is to use engineering principles to incite and promote the natural healing process of bone which does not occur in critical-sized defects. A synthetic bone scaffold must be biocompatible, biodegradable to allow native tissue integration, and mimic the multidimensional hierarchical structure of native bone. In addition to being physically and chemically biomimetic, an ideal scaffold is capable of eluting bioactive molecules (e.g., BMPs, TGF-betas, etc., to accelerate extracellular matrix production and tissue integration) or drugs (e.g., antibiotics, cisplatin, etc., to prevent undesired biological response such as sepsis or cancer recurrence) in a temporally and spatially controlled manner. Various biomaterials including ceramics, metals, polymers, and composites have been investigated for their potential as bone scaffold materials. However, due to their tunable physiochemical properties, biocompatibility, and controllable biodegradability, polymers have emerged as the principal material in bone tissue engineering. This article briefly reviews the physiological and anatomical characteristics of native bone, describes key technologies in mimicking the physical and chemical environment of bone using synthetic materials, and provides an overview of local drug delivery as it pertains to bone tissue engineering is included.