Photonic monitoring of chitosan nanostructured alginate microcapsules for drug release

By using a novel microfluidic set-up for drug screening applications, this study examines delivery of a novel risedronate based drug formulation for treatment of osteoporosis that was developed to overcome the usual shortcomings of risedronate, such as its low bioavailability and adverse gastric effects. Risedronate nanoparticles were prepared using muco-adhesive polymers such as chitosan as matrix for improving the intestinal cellular absorption of risedronate and also using a gastric-resistant polymer such as sodium alginate for reducing the gastric inflammation of risedronate. The in-vitro characteristics of the alginate encapsulated chitosan nanoparticles are investigated, including their stability, muco-adhesiveness, and Caco-2 cell permeability. Fluorescent markers are tagged with the polymers and their morphology within the microcapsules is imaged at various stages of drug release.

Imaging Flow Cytometry With Femtosecond Laser-Micromachined Glass Microfluidic Channels

Microfluidic/optofluidic microscopy is a versatile modality for imaging and analyzing properties of cells/particles while they are in flow. In this paper, we demonstrate the integration of fused silica microfluidics fabricated using femtosecond laser machining into optofluidic imaging systems. By using glass for the sample stage of our microscope, we have exploited its superior optical quality for imaging and bio-compatibility. By integrating these glass microfluidic devices into a custom-built bright field microscope, we have been able to image red blood cells in flow with high-throughputs and good fidelity. In addition, we also demonstrate imaging as well as detection of fluorescent beads with these microfluidic devices.

Targeted Modifications in Adeno-Associated Virus Serotype 8 Capsid Improves Its Hepatic Gene Transfer Efficiency In Vivo

Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T -> Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S -> A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (similar to 9- to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector bio-distribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8 mutant in therapeutic gene transfer, we delivered human coagulation factor IX (h. FIX) under the control of liver-specific promoters (LP1 or hAAT) into C57BL/6 mice. The circulating levels of h. FIX: Ag were higher in all the K137R-AAV8 treated groups up to 8 weeks post-hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel AAV8 vectors for potential gene therapy of hemophilia B.

A rapid method to assess reactive oxygen species in yeast using H2DCF-DA

A protocol to efficiently assess Reactive Oxygen Species (ROS) levels in yeast cells using H2DCF-DA is described here. This method employs lithium acetate to permeate the cell wall, and thus, augments the release of the fluorescent product, dichlorofluorescein from the cells. This protocol obviates the need for both physical and enzymatic lysis methods that are arduous and time consuming. This method is simple, less time consuming and reproducible, especially while dealing with a large sample size. The lithium acetate method gave significantly reproducible and linear results (P < 0.0001), as compared with direct measurement (P = 0.0005), sonication (P = 0.1466) and bead beating (P = 0.0028).

Dispersible crystalline nanobundles of YPO4 and Ln (Eu, Tb)-doped YPO4: rapid synthesis, optical properties and bio-probe applications

Undoped and Ln(3+) (Eu and Tb)-doped crystalline nanobundles of YPO4 were prepared by a facile microwave-assisted route with water as a solvent and without using any surfactant. TEM investigations reveal that the as-prepared powder consists of lenticular-shaped nanobundles (similar to 100 nm in diameter) made of very small nanorods with diameter less than 10 nm and length varying from 20 to 50 nm. Each nanorod in turn is single crystalline, as revealed by HRTEM imaging. The as-prepared nanobundles are easily dispersible in various solvents, especially water, without any surface functionalization, which is critical for various bio-probe applications like cell and tissue imaging. The Eu- and Tb-doped YPO4 nanobundles show good photoluminescence properties and were further evaluated for their use as fluorescent biolabels. Our results show that HeLa cells labelled with Eu- and Tb-doped YPO4 nanobundles show bright red (Eu) and green (Tb) intracellular luminescence under a confocal microscope. Concentration-and time-dependent MTT cell viability assays show that the nanobundles show low toxicity towards cells which makes them promising in bioimaging field.

pi-Electron rich small molecule sensors for the recognition of nitroaromatics

Selective and discriminative detection of -NO2 containing high energy organic compounds such as picric acid (PA), 2,4,6-trinitrotoluene (TNT) and dinitrotoluene (DNT) has become a challenging task due to concerns over national security, criminal investigations and environment protections. Among various known detection methods, fluorescence techniques have gained special attention in recent time. A wide variety of fluorescent chemosensors have been developed for nitroaromatic explosive detection. In this review article, we provide an overview of the recent developments made in small molecule-based turn-off fluorescent sensors for nitroaromatic explosives with special focus on organic and H-bonded supramolecular sensors. The fluorescent sensors discussed in this review are classified and organized according to their functionality and their recognition of nitroaromatics by fluorescence quenching.

Plasmonic Lipid Bilayer Membranes for Enhanced Detection Sensitivity of Biolabeling Fluorophores

Plasmonics based sensing, using the surface plasmon resonance of metal nanoparticles, has been effectively demonstrated in various applications. Extending this methodology to cell and artificial lipid bilayer membranes is extremely beneficial in enhancing the sensitivity of the detection of binding and cellular transport of molecules across such membranes. Here, the creation of an artificial plasmonic biomembrane template is demonstrated and used to show the enhanced detection sensitivity of certain widely used biomarker molecules. The efficacy of these templates is explained in terms of the ability of the hydrophobic polymer grafted gold nanoparticles used to organize, penetrate, and fluidize the membranes. The enhancement of photoluminescence of the dye molecules used occurs over a reasonably large spectral range as compared to the plasmon resonance of gold nanoparticles. The results could, possibly, be extended to cellular membranes with relevant modifications, as well as to the detection of any other biological molecule appropriately labeled with fluorescent dye molecules, and demonstrate the versatility of these plasmonic bioinspired platforms as potential biochemical sensors.

Targeting human telomeric G-quadruplex DNA with curcumin and its synthesized analogues under molecular crowding conditions

The formation of telomeric G-quadruplexes has been shown to inhibit telomerase activity. Indeed, a number of small molecules capable of p-stacking with G-tetrads have shown the ability to inhibit telomerase activity through the stabilization of G-quadruplexes. Curcumin displays a wide spectrum of medicinal properties ranging from anti-bacterial, anti-viral, anti-protozoal, anti-fungal and anti-inflammatory to anti-cancer activity. We have investigated the interactions of curcumin and its structural analogues with the human telomeric sequence AG(3)(T(2)AG(3))(3) under molecular crowding conditions. Experimental studies indicated the existence of a AG(3)(T(2)AG(3))(3)/curcumin complex with binding affinity of 0.72 x 10(6) M-1 under molecular crowding conditions. The results from UV-visible absorption spectroscopy, a fluorescent TO displacement assay, circular dichroism and molecular docking studies, imply that curcumin and their analogues interact with G-quadruplex DNA via groove binding. While other analogs of curcumin studied here bind to G-quadruplexes in a qualitatively similar manner their affinities are relatively lower in comparison to curcumin. The Knoevenagel condensate, a methoxy-benzylidene derivative of curcumin, also exhibited significant binding to G-quadruplex DNA, although with two times decreased affinity. Our study establishes the potential of curcumin as a promising natural product for G-quadruplex specific ligands.

Targeting human telomeric G-quadruplex DNA with curcumin and its synthesized analogues under molecular crowding conditions

The formation of telomeric G-quadruplexes has been shown to inhibit telomerase activity. Indeed, a number of small molecules capable of p-stacking with G-tetrads have shown the ability to inhibit telomerase activity through the stabilization of G-quadruplexes. Curcumin displays a wide spectrum of medicinal properties ranging from anti-bacterial, anti-viral, anti-protozoal, anti-fungal and anti-inflammatory to anti-cancer activity. We have investigated the interactions of curcumin and its structural analogues with the human telomeric sequence AG(3)(T(2)AG(3))(3) under molecular crowding conditions. Experimental studies indicated the existence of a AG(3)(T(2)AG(3))(3)/curcumin complex with binding affinity of 0.72 x 10(6) M-1 under molecular crowding conditions. The results from UV-visible absorption spectroscopy, a fluorescent TO displacement assay, circular dichroism and molecular docking studies, imply that curcumin and their analogues interact with G-quadruplex DNA via groove binding. While other analogs of curcumin studied here bind to G-quadruplexes in a qualitatively similar manner their affinities are relatively lower in comparison to curcumin. The Knoevenagel condensate, a methoxy-benzylidene derivative of curcumin, also exhibited significant binding to G-quadruplex DNA, although with two times decreased affinity. Our study establishes the potential of curcumin as a promising natural product for G-quadruplex specific ligands.

White light emissive molecular siblings

The design and synthesis of two structurally close and complementarily fluorescent boron based molecular siblings 2 and 3 are reported. The luminescence properties of individual triads are modulated to complement each other by controlling the intramolecular energy transfer in 2 and 3. The binary mixture of 2 and 3 emits white-light.