Spectroscopic features of a perylenediimide probe for sensing amyloid fibrils: in vivo imaging of Aß-aggregates in a Drosophila model organism.

Customised perylenediimide (PDI) chromophores find diverse applications not only as chemosensors, inorganic-organic semiconductors, photovoltaics, photocatalysts, etc., but also in protein surface engineering, bio-sensors and drug delivery systems. This study focuses on the interaction of a custom synthesized phenylalanine derivatized perylenediimide (L-Phe-PDI) dye with a model protein, insulin, and its structurally distinct fibrils to develop fluorescence sensors for fibrillar aggregates and in vivo imaging applications. Detailed photophysical studies revealed that L-Phe-PDI gets aggregated in the presence of insulin and causes emission quenching at pH 7.4, which in the absence of insulin occurs only at pH ∼2. During in vitro incubation of insulin to its fibrils, the fluorescence intensity of the L-Phe-PDI probe is enhanced to ∼150 fold in a two-stage manner, manifesting the pathways of structural transformation to ß-sheet rich mature fibrils. The in vivo sensing has further been validated in living models of the Aß-mutant Drosophila fly, which is known to develop progressive neurodegeneration comparable to that of human brains with Alzheimer's disease (AD). Bioimaging of the L-Phe-PDI treated Aß-mutant Drosophila documented the blood-brain/blood-retina-barrier cross-over ability of L-Phe-PDI with no toxic effects. Comparison of the fibrillar images from the brain and eye region with the reference thioflavin T (ThT) probe established the uptake of L-Phe-PDI by the aggregate/fibrillar moieties. The samples from L-Phe-PDI-treated flies apparently displayed reduced fibrillar spots, a possible case of L-Phe-PDI-induced disintegration of fibrillar aggregates at large, an observation substantiated by the improved phenotype activities as compared to the untreated flies. The findings reported both in vitro and in vivo with the L-Phe-PDI material for the first time open up avenues to explore the therapeutic potential of custom-designed PDI derivatives for amyloid fibril sensors and bioimaging.

A quick and laidback way to detect the internal structure of the Drosophila eye: An alternative to cryosectioning.

Immunofluorescence techniques have been a great tool to chase the structure, localization, and function of many proteins within a cell. Drosophila eye is widely used as a model to answer various questions. However, the complex sample preparation and visualization methods restrict its use only with an expert's hand. Thus, an easy and hassle-free method is in need to broaden the use of this model even with an amateur's hand. The current protocol describes an easy sample preparation method using DMSO to image the adult fly eye. The brief description of sample collection, preparation, dissection, staining, imaging, storage, and handling has been described over here. For readers, the possible problems that might arise during the execution of the experiment have been described with their possible reason and solutions. The overall protocol reduces the use of chemicals and shortens the sample preparation time to only 3 h, which is significantly less in comparison to other protocols.

Synthesis and Characterizations of Bioactive Glass Nanoparticle-Incorporated Triblock Copolymeric Injectable Hydrogel for Bone Tissue Engineering.

Recently, injectable hydrogels have attracted much interest in tissue engineering (TE) applications because of their controlled flowability, adaptability, and easy handling properties. This work emphasizes the synthesis and characterizations of bioactive glass (BAG) nanoparticle-reinforced poly(ethylene glycol) (PEG)- and poly(N-vinylcarbazole) (pNVC)-based minimally invasive composite injectable hydrogel suitable for bone regeneration. First, the copolymer was synthesized from a combination of PEG and pNVC through reversible addition-fragmentation chain-transfer (RAFT) polymerization and nanocomposite hydrogel constructs were subsequently prepared by conjugating BAG particles at varying loading concentrations. Gel permeation chromatography (GPC) analysis confirmed the controlled nature of the polymer. Various physicochemical characterization results confirmed the successful synthesis of copolymer and nanocomposite hydrogels that showed good gelling and injectability properties. Our optimal nanocomposite hydrogel formulation showed excellent swelling properties in comparison to the copolymeric hydrogel due to the presence of hydrophilic BAG particles. The bone cell proliferation rate was found to be evidently higher in the nanocomposite hydrogel than in the copolymeric hydrogel. Moreover, the enhanced level of ALP activity and apatite mineralization for the nanocomposite in comparison to that for the copolymeric hydrogel indicates accelerated in vitro osteogenesis. Overall, our study findings indicate BAG particle-conjugated nanocomposite hydrogels can be used as promising grafting materials in orthopedic reconstructive surgeries complementary to conventional bone graft substitutes in cancellous bone defects due to their 3D porous framework, minimal invasiveness, and ability to form any desired shape to match irregular bone defects.

Fibril-induced neurodegenerative disorders in an Aß-mutant Drosophila model: therapeutic targeting using ammonium molybdate.

The ability of polyanionic molybdate to inhibit and degrade protein fibrils both in vitro (insulin protein) and in vivo (Drosophila fly model) has been demonstrated. We establish the disappearance of fibrillar structures and recovery from neurodegenerative disorders in molybdate-treated Aß42-mutant Drosophila flies as compared to the untreated ones, corroborating the therapeutic ability of ammonium molybdate towards the treatment of Alzheimer's disease.

Dextran based amphiphilic self-assembled biopolymeric macromolecule synthesized via RAFT polymerization as indomethacin carrier.

This work demonstrates a facile pathway to develop a biopolymer based amphiphilic macromolecule through reversible addition-fragmentation chain transfer (RAFT) polymerization, using dextran (a biopolymer) as starting material. Also, a new hydrophobic monomer [2-methyl-acrylic acid 1-benzyl-1H-[1,2,3] triazol-4-ylmethyl ester (MABTE)] has been synthesized using methacrylic acid via "click" approach. The resultant copolymer displays controlled radical polymerization characteristics: narrow polydispersity (Ð) and controlled molecular weight as obtained through advanced polymer chromatography (APC) analysis. In aqueous solution, the copolymer can proficiently be self-assembled to provide micellar structure, which has been evidenced from field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) analyses. The in-vitro cytotoxicity study illustrates the nontoxic nature of the copolymer up to 100 µg/mL polymer concentration. The copolymer has been found to be worthy as an efficient carrier for the sustained release of hydrophobic drug: Indomethacin (IND).

Amphiphilic graft copolymeric micelle using dextrin and poly (N-vinyl caprolactam) via RAFT polymerization: Development and application.

Dextrin and poly (N-vinyl caprolactam) based amphiphilic graft copolymer has recently been developed using RAFT polymerization. The prepared graft copolymer has been characterized in details using FTIR and 1H NMR spectral analyses, GPC, TGA, FESEM, TEM and DLS analyses. GPC analysis results indicate that the polymerization is controlled, while the LCST value of the copolymer suggests that the synthesized copolymer demonstrates sol-gel behaviour on applying temperature. FESEM and TEM analyses envisage that the graft copolymer has spherical morphology with nanoscale dimension. DLS study reveals that the micellar size vary with change of pH and temperature, demonstrating the dual-responsive behaviour of the graft copolymer. Finally, the developed graft copolymer shows worthy efficacy towards the pH and thermo-responsive release of encapsulated pyrene in sustained manner.