Development of Cell Culture Platforms for Study of Trabecular Meshwork Cells and Glaucoma Development.

BACKGROUND: Various cell culture platforms that could display native environmental cue-mimicking stimuli were developed, and effects of environmental cues on cell behaviors were studied with the cell culture platforms. Likewise, various cell culture platforms mimicking native trabecular meshwork (TM) composed of juxtacanalicular, corneoscleral and uveal meshwork located in internal scleral sulcus were used to study effects of environmental cues and/or drug treatments on TM cells and glaucoma development. Glaucoma is a disease that could cause blindness, and cause of glaucoma is not clearly identified yet. It appears that aqueous humor (AH) outflow resistance increased by damages on pathway of AH outflow can elevate intraocular pressure (IOP). These overall possibly contribute to development of glaucoma. METHODS: For the study of glaucoma, static and dynamic cell culture platforms were developed. Particularly, the dynamic platforms exploiting AH outflow-mimicking perfusion or increased IOP-mimicking increased pressure were used to study how perfusion or increased pressure could affect TM cells. Overall, potential mechanisms of glaucoma development, TM structures and compositions, TM cell culture platform types and researches on TM cells and glaucoma development with the platforms were described in this review. RESULTS AND CONCLUSION: This will be useful to improve researches on TM cells and develop enhanced therapies targeting glaucoma.

Efficient Combination Chemo-Sonodynamic Cancer Therapy Using Mitochondria-Targeting Sonosensitizer-Loaded Polysorbate-Based Micelles.

Sonodynamic therapy (SDT), utilizing ultrasound (US) and sonosensitizers, holds immense potential as a noninvasive and targeted treatment for a variety of deep-seated tumors. However, the clinical translation of SDT is hampered by several key limitations in sonosensitizers, especially their low aqueous stability and poor cellular uptake. In this study, non-ionic polysorbate (Tween 80, T80) was adopted to formulate effective nanocarriers for the safe and efficient delivery of sonosensitizers to cancer cells. Mitochondria-targeting triphenylphosphonium (TPP)-conjugated chlorin e6 (Ce6) sonosensitizer was loaded into T80-based micelles for efficient SDT. Pro-oxidant piperlongumine (PL) was co-encapsulated with TPP-conjugated Ce6 (T-Ce6) in T80 micelles to enable combination chemo-SDT. T80 micelles substantially enhanced the cellular internalization of T-Ce6. As a result, T80 micelles loaded with T-Ce6 and PL [T80(T-Ce6/PL)] significantly elevated intracellular reactive oxygen species (ROS) generation in MCF-7 human breast cancer cells upon US exposure. Moreover, T-Ce6 exhibited selective accumulation within the mitochondria, leading to efficient cell death under US irradiation. Importantly, T80(T-Ce6/PL) micelles caused cancer-specific cell death by selectively triggering apoptosis in cancer cells through PL. This study demonstrated the feasibility of using T80(T-Ce6/PL) micelles for efficient and cancer-specific combination chemo-SDT.

Processing DNA Storage through Programmable Assembly in a Droplet-Based Fluidics System.

DNA can be used to store digital data, and synthetic short-sequence DNA pools are developed to store high quantities of digital data. However, synthetic DNA data cannot be actively processed in DNA pools. An active DNA data editing process is developed using splint ligation in a droplet-controlled fluidics (DCF) system. DNA fragments of discrete sizes (100-500 bps) are synthesized for droplet assembly, and programmed sequence information exchange occurred. The encoded DNA sequences are processed in series and parallel to synthesize the determined DNA pools, enabling random access using polymerase chain reaction amplification. The sequencing results of the assembled DNA data pools can be orderly aligned for decoding and have high fidelity through address primer scanning. Furthermore, eight 90 bps DNA pools with pixel information (png: 0.27-0.28 kB), encoded by codons, are synthesized to create eight 270 bps DNA pools with an animation movie chip file (mp4: 12 kB) in the DCF system.

De novo generation of a bright blue fluorophore from 2-oxoglutarate in biological samples.

We discovered the generation of a new bright blue fluorophore from a particular type of amine and 2-oxoglutarate (2-OG) under mild conditions without any chemical additives. Two ß-aminoethylamine molecules and three 2-OG molecules form an unprecedented 2-pyridone structure with a fused γ-lactam ring (DTPP) via complex reactions including double decarboxylation and quintuple dehydration. The DTPP fluorophore shows a high quantum yield (80%) and photostability. The great potential of the present DTPP generation in the quantitative analysis of 2-OG in biosamples is demonstrated.

Programmable DNA-Based Boolean Logic Microfluidic Processing Unit.

As molecular computing materials, information-encoded deoxyribonucleic acid (DNA) strands provide a logical computing process by cascaded and parallel chain reactions. However, the reactions in DNA-based combinational logic computing are mostly achieved through a manual process by adding desired DNA molecules in a single microtube or a substrate. For DNA-based Boolean logic, using microfluidic chips can afford automated operation, programmable control, and seamless combinational logic operation, similar to electronic microprocessors. In this paper, we present a programmable DNA-based microfluidic processing unit (MPU) chip that can be controlled via a personal computer for performing DNA calculations. To fabricate this DNA-based MPU, polydimethylsiloxane was cast using double-sided molding techniques for alignment between the microfluidics and valve switch. For a uniform surface, molds fabricated using a three-dimensional printer were spin-coated by a polymer. For programming control, the valve switch arms were operated by servo motors. In the MPU controlled via a personal computer or smartphone application, the molecules with two input DNAs and a logic template DNA were reacted for the basic AND and OR operations. Furthermore, the DNA molecules reacted in a cascading manner for combinational AND and OR operations. Finally, we demonstrated a 2-to-1 multiplexer and the XOR operation with a three-step cascade reaction using the simple DNA-based MPU, which can perform Boolean logic operations (AND, OR, and NOT). Through logic combination, this DNA-based Boolean logic MPU, which can be operated using programming language, is expected to facilitate the development of complex functional circuits such as arithmetic logical units and neuromorphic circuits.

Development of poly(D,L-lactic-co-glycolic acid) films coated with biomembrane-mimicking polymers for anti-adhesion activity.

A physical barrier is one of the most effective strategies to alleviate excessive postoperative adhesion (POA) between tissues at an injury site. To overcome the limitations of current polymeric film-type physical barriers, we suggest a film of poly(lactic-co-glycolic acid) (PLGA) that is non-covalently coated with poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)) (PMB). While maintaining the degradability and mechanical properties of PLGA, the PMB coating introduces strong anti-adhesive properties to the film by forming a zwitterionic MPC-based surface through the hydrophobic interactions between BMA moieties and PLGA. Compared to SurgiWrap®, the commercially available poly(lactic acid)-based anti-adhesive film against POA, the PMB-coated PLGA film is much more inhibitory against protein adsorption and fibroblast adhesion, processes that are crucial to the POA process. PMB coating also inhibits the expression of fibronectin containing extra domain A (FN-EDA), α-smooth muscle actin (α-SMA), and collagen type IV alpha 2 (COL4A2), which are marker genes and proteins involved in fibroblast activation and excessive fibrosis during POA. Such inhibitory activities are clearly observed in a 3-dimensional culture of fibroblasts within a collagen matrix, which mimics the in vivo environment of an injury site, as well as in a 2-dimensional culture. The kinetics and the stability of the PMB coating suggest potential future clinical use to coat PLGA films to create a film-type anti-adhesion barrier that overcomes the limitations of current products.

Helicity Control of Polymeric Backbones with Alternating cis-trans Double Bonds in Cyclopolymerized Dipropargyl Amides.

We report the synthesis of new helical polymeric structures having alternating cis and trans double bonds and chiral amino acid side chains by metathesis cyclopolymerization. The polymer helicity, which is generated by the interaction between fluorenylmethyloxycarbonyl (Fmoc) groups in the side chains, is dramatically affected by solvents. A thorough experimental and theoretical analysis including nuclear magnetic resonance, atomic force microscopy, and density functional theory and molecular mechanics calculations suggests that the helicity of both backbone and side chains are determined by anti-syn rotation of the carbamate groups and by the different interactions of the Fmoc groups with solvents.

The poly-thymine based DNA photolithography onto electrostatic coupling substrates.

In order to develop a rapid and high fidelity process for DNA self-assembly with patterning, the pattern of thymine dimerization is presented onto electrostatically bound DNA substrate by photolithography. The ability of binding for the process, which is attenuated conditions such as contact with photomask and washing by solution buffer is evaluated by X-ray photoelectron spectroscopy (XPS). Through thymine dimerization and hybridization chain reaction (HCR), DNA patterns, including multi-patterns, are demonstrated. For expansion to protein molecular patterning, the target DNA is tethered to biotin, allowing patterning with streptavidin linked fluorophores such as Cy3-streptavidin and phyecocayine-streptavidin.

α-Helical cell-penetrating peptide-mediated nasal delivery of resveratrol for inhibition of epithelial-to-mesenchymal transition.

In the present study, we examined the potential of cell-penetrating peptide (CPP)-based intranasal drug delivery for the treatment of localized nasal diseases. Many charged or non-hydrophobic drugs have difficulty penetrating into the nasal epithelium due to intrinsic membrane impermeability and rapid mucociliary clearance in the nasal cavity. To treat chronic rhinosinusitis with nasal polyps (CRSwNP), one of the most common localized nasal diseases, we conjugated resveratrol (RSV) to an amphiphilic α-helical leucine (L)- and lysine (K)-rich CPP (LK) and intranasally delivered it to the interior of nasal epithelial cells for inhibiting epithelial-to-mesenchymal transition (EMT) caused by hypoxia-inducible factor 1α. The RSV-LK conjugate could penetrate into the nasal epithelium and efficiently inhibit EMT, nasal polyp formation, epithelial disruption, and related inflammation in an eosinophilic CRSwNP mouse model, at 10-fold lower doses and with 3-fold less frequent administration than free RSV. Due to the rapid penetration into the nasal epithelium and the therapeutic effect of the RSV-LK conjugate at much lower doses than free RSV, this CPP-based delivery system, with the ability to overcome the tight nasal epithelial barrier, may provide a new strategy for the treatment of localized nasal diseases without the systemic side effects of cargo drugs.

ZnO Nanocluster-Functionalized Single-Walled Carbon Nanotubes Synthesized by Microwave Irradiation for Highly Sensitive NO2 Detection at Room Temperature.

To improve the NO2-sensing performance of single-walled carbon nanotube (SWCNT)-based sensors, zinc oxide (ZnO) nanoclusters (NCs) were functionalized by a microwave (MW)-assisted synthesis technique. Gas sensors based on pristine SWCNTs and ZnO NC-SWCNT composites synthesized using different weight ratios (ZnO/SWCNTs = 0.5:1, 1:1, 2:1, and 3:1) were fabricated, and their ability to sense various gases at room temperature (25 °C) was investigated. The results showed that the sensing performance of the ZnO NC-SWCNT composite synthesized with a weight ratio of 1:1 (denoted as Z-SWCNTs) was significantly enhanced with respect to NO2 response and selectivity. This enhanced sensing performance is thought to be a result of both the modulation of the conduction channel at the ZnO NC-SWCNT heterointerfaces and the generation of defects (or holes) by MW irradiation that act as active sites for the target gases. The results obtained in this work provide not only a facile method of cofunctionalizing oxide NCs and defects but also a new methodology for improving the sensing capabilities of SWCNT-based gas sensors.

Calcium-Binding Polymer-Coated Poly(lactide- co-glycolide) Microparticles for Sustained Release of Quorum Sensing Inhibitors to Prevent Biofilm Formation on Hydroxyapatite Surfaces.

Quorum sensing (QS) inhibitor-based therapy is an attractive strategy to inhibit bacterial biofilm formation without excessive induction of antibiotic resistance. Thus, we designed Ca2+-binding poly(lactide- co-glycolide) (PLGA) microparticles that can maintain a sufficient concentration of QS inhibitors around hydroxyapatite (HA) surfaces in order to prevent biofilm formation on HA-based dental or bone tissues or implants and, therefore, subsequent pathogenesis. Poly(butyl methacrylate- co-methacryloyloxyethyl phosphate) (PBMP) contains both Ca2+-binding phosphomonoester groups and PLGA-interacting butyl groups. The PBMP-coated PLGA (PLGA/PBMP) microparticles exhibited superior adhesion to HA surfaces without altering the sustained release properties of uncoated PLGA microparticles. PLGA/PBMP microparticle-encapsulating furanone C-30, a representative QS inhibitor, effectively inhibited the growth of Streptococcus mutans and its ability to form biofilms on HA surface for prolonged periods of up to 100 h, which was much longer than either furanone C-30 in its free form or when encapsulated in noncoated PLGA microparticles.

Ring Opening Metathesis Polymerization of Bicyclic α,ß-Unsaturated Anhydrides for Ready-to-be-grafted Polymers Having Tailored pH-Responsive Degradability.

Polymers having α,ß-unsaturated anhydrides as repeating units were synthesized by ring opening metathesis polymerization (ROMP). The anhydride moieties were ready-to-be-grafted with amines to form acid-labile cis-α,ß-unsaturated acid amide linkages. The pH-responsive reversible de-grafting can be controlled by changing the intramolecular accessibility between acid and amide groups. The alendronate-grafted ROMP polymers showed distinct pH-dependent cytotoxicity according to the anhydride structures.

DNA multi-bit non-volatile memory and bit-shifting operations using addressable electrode arrays and electric field-induced hybridization.

DNA has been employed to either store digital information or to perform parallel molecular computing. Relatively unexplored is the ability to combine DNA-based memory and logical operations in a single platform. Here, we show a DNA tri-level cell non-volatile memory system capable of parallel random-access writing of memory and bit shifting operations. A microchip with an array of individually addressable electrodes was employed to enable random access of the memory cells using electric fields. Three segments on a DNA template molecule were used to encode three data bits. Rapid writing of data bits was enabled by electric field-induced hybridization of fluorescently labeled complementary probes and the data bits were read by fluorescence imaging. We demonstrated the rapid parallel writing and reading of 8 (23) combinations of 3-bit memory data and bit shifting operations by electric field-induced strand displacement. Our system may find potential applications in DNA-based memory and computations.

An Implantable Transparent Conductive Film with Water Resistance and Ultrabendability for Electronic Devices.

Recently, instead of indium tin oxide, the random mesh pattern of metallic nanowires for flexible transparent conducting electrodes (FTCEs) has received a great amount of interest due to its flexibility, low resistance, reasonable price, and compliant processes. Mostly, nanowires for FTCEs are fabricated by spray or mayer coating methods. However, the metallic nanowire layer of FTCEs, which is fabricated by these methods, has a spiked surface roughness and low junction contact between the nanowires that lead to their high sheet resistance value. Also, the nanowires on FTCEs are easy to peel-off through exterior forces such as bending, twisting, or contact. To solve these problems, we demonstrate novel methods through which silver nanowires (AgNWs) are deposited onto a nanosize porous nitrocellulose (NC) substrate by electrophoretic deposition (EPD) and an opaque and porous substrate. Respectively, through dimethyl sulfoxide treatment, AgNWs on NC (AgNW/NC) is changed to the transparent and nonporous FTCEs. This enhances the junction contact of the AgNWs by EPD and also allows a permanent attachment of AgNWs onto the substrate. To show the mechanical strength of the AgNWs on the transparent nitrocellulose (AgNW/TNC), it is tested by applying diverse mechanical stress, such as a binding test (3M peel-off), compressing, bending, twisting, and folding. Next, we demonstrate that AgNW/TNC can be effectively implanted onto normal newspapers and papers. As paper electronics, light-emitting diodes, which are laminated onto paper, are successfully operated through a basic AgNW/TNC strip circuit. Finally, it is demonstrated that AgNW/TNC and AgNW/TNC on paper are water resistant for 15 min due to the insulation properties of the nonporous substrate.

Polymorphic Architectures of Graphene Quantum Dots.

A systematic strategy for designing structured nanomaterials is demonstrated through self-assembly of graphene quantum dots. The approach reveals that graphene derivatives at the nanoscale assemble into various architectures of nanocrystals in a binary solution system. The shapes of the nanocrystals continue to evolve in terms of the intimate association of organic molecules with the dispersion medium, obtaining a high index faceted superlattice. This facile synthetic process provides a versatile strategy for designing particles to new structured materials systems, exploiting the crystallization of layered graphitic carbon structures within single crystals.

Amine-selective affinity resins based on pH-sensitive reversible formation of covalent bonds.

A new class of affinity resins using reversible covalent bonds is introduced for the separation of amine-containing molecules. pH-sensitive reversible formation of amic acid bonds between amines and carboxylate dimethyl maleic anhydride-decorated wrinkled silica nanoparticle resins was used to selectively retain and release amine-containing molecules, by controlling the pH.

A Programmable DNA Double-Write Material: Synergy of Photolithography and Self-Assembly Nanofabrication.

We demonstrate a DNA double-write process that uses UV to pattern a uniquely designed DNA write material, which produces two distinct binding identities for hybridizing two different complementary DNA sequences. The process requires no modification to the DNA by chemical reagents and allows programmed DNA self-assembly and further UV patterning in the UV exposed and nonexposed areas. Multilayered DNA patterning with hybridization of fluorescently labeled complementary DNA sequences, biotin probe/fluorescent streptavidin complexes, and DNA patterns with 500 nm line widths were all demonstrated.

An Aqueous Single Reactor Arc Discharge Process for the Synthesis of Graphene Nanospheres.

Using an aqueous single reactor arc discharge process with oil-in-water emulsions allows production of 2D multilayered graphenes (MLGs and 3D graphene-based crumpled/sphere-like particles with low levels of defects). The confinement forces to create 3D particles from 2D MLGs are estimated to be 2.5 µN for crumpled particles and 70 µN for spherical hollow particles.

Device for dielectrophoretic separation and collection of nanoparticles and DNA under high conductance conditions.

Most dielectrophoretic (DEP) separations of cells, nanoparticles, and other entities are carried out on microelectrode arrays or in microfluidic device formats. Less work has been directed at designing pipette-type formats that would allow dipping into and recovering specific analytes from samples in microtiter plate formats. In order to address this important area, we have fabricated micropipette tip devices containing a 2% agarose gel plug, a buffer chamber, and platinum electrode as the DEP collection device, to be used in combination with separate sample wells that contain a circular gold electrode. We demonstrated that 200 nm fluorescent nanoparticles could be isolated into DEP high-field regions and separated from 10 µm fluorescent microbeads in high conductance buffer (1× PBS) by applying an alternating current at 10 kHz with a peak-to-peak voltage (Vpp) of 160 Vpp. The collected nanoparticles were then transferred to a new buffer solution. We also demonstrated the DEP isolation and separation of genomic DNA (>50 kbps) from the 10 µm microbeads in high conductance buffer (1× PBS) with transfer of collected DNA to another solution.

An Electric Field Assembler System for Micro-Nanofabrication of Energy Storage Materials.

Traditional methods have limitations for the fabrication of micro-ordered batteries for use in a variety of applications including biomedical and healthcare devices. A new micro-nanofabrication method and system that combines deposition, layering, and patterning processes has now been developed for production of micro-ordered energy storage battery devices and materials. Here, we show an electrical field directed (EFD) deposition array device with a 500 µm active assembly area containing 25 microelectrodes. Using EFD assembly, we demonstrated the patterned deposition of graphite mixtures in water-based and solvent-based solutions. The graphite mixture patterns were confirmed by SEM imaging.