Structural and functional characterization of proteins adsorbed on hydrophilized polylactide-co-glycolide microfibers

Rajesh Vasita, Dhirendra S KattiDepartment of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, Uttar Pradesh, IndiaBackground: Hydrophobic biopolymers such as polylactide-co-glycolide (PLGA, 85:15) have been extensively explored as scaffolding materials for tissue engineering applications. More recently, electrospun microfiber-based and nanofiber-based scaffolds of PLGA have received increased attention because they act as physical mimics of the fibrillar extracellular matrix. However, the hydrophobicity of the PLGA microfiber surface can limit its use in biomedical applications. Therefore, in a previous study, we fabricated Pluronic® F-108 (PF-108)-blended PLGA microfibrous scaffolds that alleviated the hydrophobicity associated with PLGA by enriching the surface of microfibers with the ethylene oxide units present in PF-108.Methods: In this study, we report the influence of the extent of surface enrichment of PLGA microfibers on their interaction with two model proteins, ie, bovine serum albumin (BSA) and lysozyme. BSA and lysozyme were adsorbed onto PLGA microfiber meshes (unmodified and modified) and studied for the amount, secondary structure conformation, and bioactivity of released protein.Results: Irrespective of the type of protein, PF-108-blended PLGA microfibers showed significantly greater protein adsorption and release than the unblended PLGA samples. However, in comparison with BSA, lysozyme showed a 7–9-fold increase in release. The Fourier transform infrared spectroscopy studies for secondary structure determination demonstrated that irrespective of type of microfiber surface (unblended or blended), adsorbed BSA and lysozyme did not show any significant change in secondary structure (α-helical content) as compared with BSA and/or lysozyme in the free powder state. Further, the bioactivity assay of lysozyme released from blended PLGA microfiber meshes demonstrated 80%–85% bioactivity, indicating that the process of adsorption did not significantly affect biological activity. Therefore, this study demonstrated that the decreased hydrophobicity of blended PLGA microfibrous meshes not only improved the amount of protein adsorbed (lysozyme and BSA) but also maintained the secondary structure and bioactivity of the adsorbed proteins.Conclusion: Modulating the hydrophobicity of PLGA via blending with PF-108 could be a viable strategy to improve its interaction with proteins and subsequent cell interaction in tissue engineering applications.Keywords: microfiber, protein adsorption, electrospinnin

Bi-functional oxidized dextran–based hydrogel inducing microtumors: An in vitro three-dimensional lung tumor model for drug toxicity assays

Cancer is a serious death causing disease having 8.2 million deaths in 2012. In the last decade, only about 10% of chemotherapeutic compounds showed productivity in drug screening. Two-dimensional culture assays are the most common in vitro drug screening models, which do not precisely model the in vivo condition for reliable preclinical drug screening. Three-dimensional scaffold–based cell cultures perhaps mimic tumor microenvironment and recapitulate physiologically more relevant tumor. This study was carried out to develop bi-functional oxidized dextran–based cell instructive hydrogel that provides three-dimensional environment to cancer cells for inducing microtumor. Oxidized dextran was blended with thiolated chitosan to fabricate an in situ self-gelable hydrogel (modified dextran–chitosan) in a one-step process. The hydrogels characterization revealed cross-linked network structure with highly porous structure and water absorption. The modified dextran–chitosan hydrogel showed reduced hydrophobicity and has reduced protein absorption, which resulted in changing the A549 cell adhesiveness, and encouraged them to form microtumor. The cells were proliferated in clusters having spherical morphology with randomly oriented stress fiber and large nucleus. Further microtumors were studied for hypoxia where reactive oxygen species generation demonstrated 15-fold increase as compared to monolayer culture. Drug-sensitivity results showed that microtumors generated on modified dextran–chitosan hydrogel showed resistance to doxorubicin with having 33%–58% increased growth than two-dimensional monolayer model at concentrations of 25–100 µM. In summary, the modified dextran–chitosan scaffold can provide surface chemistry that induces three-dimensional microtumors with physiologically relevant properties to in vivo tumor including growth, morphology, extracellular matrix production, hypoxic phenotype, and drug response. This model can be potentially utilized for drug toxicity studies and cancer disease modeling to understand tumor phenotype and progression

Oxidation of phenothiazine based fluorescent probe for hypochlorite and its application to live cell imaging

New phenothiazine appended BODIPY derivative has been synthesized and characterized for the determination of hypochlorite in live cells. The fluorescent probe has exhibited 72 fold fluorescence enhancement upon addition of hypochlorite over other reactive oxygen species. Further, the studies proved that the probe has ability to detect the hypochlorite even at extremely low level (4.1 nM). Having less cytotoxicity and negligible auto-fluorescence, the probe was successfully used to monitor external addition and internal secretion of hypochlorite in RAW 264.7 cells.Mani Vedamalaia, Dhaval Kedaria, Rajesh Vasita and Iti Gupt

Scanning electron micrograph of poly(l-lactic acid) (PLLA) nanofibrous foam synthesized from 2

5% (wt/v) PLLA/tetrahydrofuran solution at a gelation temperature of 8°C using the phase separation technique (image 500 ×). Source: Ma PX, Zhang R. 1998. Synthetic nano-scale fibrous extracellular matrix. , 46:60–72. Copyright © 1998 J Wiley. Reprinted with permission of John Wiley&Sons Inc.<p><b>Copyright information:</b></p><p>Taken from "Nanofibers and their applications in tissue engineering"</p><p></p><p>International Journal of Nanomedicine 2006;1(1):15-30.</p><p>Published online Jan 2006</p><p>PMCID:PMC2426767.</p><p>© 2006 Dove Medical Press Limited. All rights reserved</p

BODIPY-based clickates for the detection Of Hg2+ Ions through PET-CHEF mechanisms and their application to bioimaging

by Vedamalai Mani, Dhaval Vasita, Rajesh and Iti Gupt

Study of locust bean gum reinforced cyst-chitosan and oxidized dextran based semi-IPN cryogel dressing for hemostatic application

Severe blood loss due to traumatic injuries remains one of the leading causes of death in emergency settings. Chitosan continues to be the candidate material for hemostatic applications due to its inherent hemostatic properties. However, available chitosan-based dressings have been reported to have an acidic odor at the wound site due to the incorporation of acid based solvents for their fabrication and deformation under compression owing to low mechanical strength limiting its usability. In the present study semi-IPN cryogel was fabricated via Schiff's base cross-linking between the polyaldehyde groups of oxidized dextran and thiolated chitosan in presence of locust bean gum (LBG) known for its hydrophilicity. Polymerization at −12 °C yielded macroporous semi-IPN cryogels with an average pore size of 124.57 ± 20.31 μm and 85.46% porosity. The hydrophobicity index of LBG reinforced semi-IPN cryogel was reduced 2.42 times whereas the swelling ratio was increased by 156.08% compare to control cryogel. The increased hydrophilicity and swelling ratio inflated the compressive modulus from 28.1 kPa to 33.85 for LBG reinforced semi-IPN cryogel. The structural stability and constant degradation medium pH were also recorded over a period of 12 weeks. The cryogels demonstrated lower adsorption affinity towards BSA. The cytotoxicity assays (direct, indirect) with 3T3-L1 fibroblast cells confirmed the cytocompatibility of the cryogels. The hemolysis assay showed <5% hemolysis confirming blood compatibility of the fabricated cryogel, while whole blood clotting and platelet adhesion assays confirmed the hemostatic potential of semi-IPN cryogel. Keywords: Chitosan, Oxidized dextran, Locust bean gum, Cryogel, Hemostasi

Design and synthesis of BODIPY-Clickates based Hg2+ sensors: effect of triazole binding mode with Hg2+ on signal transduction

BODIPY-Clickates, F1 and F2, for the detection of Hg2+ have been designed, synthesized and characterized. Both F1 and F2 showed hyperchromic shifts with bathochromic shifts in the UV-visible spectra in response to increasing Hg2+ concentrations. Hg2+ ion binding caused perturbation of photo-induced electron transfer and chelation induced enhanced bathochromic emission of F1 and F2 to 620 nm and 660 nm, respectively. Job plot clearly indicated that binding ratio of F1 and F2 with Hg2+ was 1:1. The NMR titration of F1 and F2 with Hg2+ confirmed that aromatic and triazole were involved in the binding event. Furthermore, HRMS data of F1.Hg2+ and F2.Hg2+ supported the formation of mercury complexes of BODIY-Clickates. Dissociation constant for the interaction between fluorescent probes F1 and F2 with Hg2+ was found to be 24.4 ± 5.1 µM and 22.0 ± 3.9 µM, respectively. Hg2+ ion induced fluorescence enhancement was almost stable in a pH range of 5 to 8. Having less toxicity to live cells, both the probes were successfully used to map the Hg2+ ions in live A549 cells.by Iti Gupta, Vedamalai Mani, Dhaval Kedaria, Rajesh Vasita and Shigeki Mori