Alginate Capsules with Cuttlebone-derived Fillers as an Integrated Solution for Bone Repair

Alginate capsules with cuttlebone-derived fillers were developed for bone repair applications. Prepared capsules were designed to be suitable for the treatment of small-sized bone loss provocative diseases, such as endodontic and periodontal diseases. Cuttlebone microparticles, as a source of calcium carbonate, or cuttlebone-derived hydroxyapatite microparticles were used as mineral fillers for the preparation of alginate capsules. The capsules were additionally covered with chitosan layer for the hard structure formation and improvement of adhesive properties. Encapsulation efficiency of dexamethasone as a model allopathic drug was 19 %, 24 % and 12 % for capsules with cuttlebone microparticles, capsules with cuttlebone-derived hydroxyapatite and capsules without any mineral filler (control group), respectively. We observed that chitosan coating and lyophilisation contributed for the preservation of spherical structure of alginate capsules with mineral fillers. Swelling study of dried capsules in simulated physiological environment (in phosphate buffer saline, pH = 7.2 at 37 °C temperature) showed that average swelling index of alginate-based capsules without mineral fillers was much higher in comparison to capsules with cuttlebone fillers: 121 %, 17 % and 3 % for control group, capsules with cuttlebone microparticles and capsules with cuttlebone-derived hydroxyapatite, respectively.DOI: http://dx.doi.org/10.5755/j01.ms.24.3.18858</p

Screening of lipase carriers for reactions in water, biphasic and pure organic solvent systems*

In bioprocesses lipases are typically used in immobilized form, irrespective of type of reaction systems, to ensure an even distribution of catalysts in water restricted media and/or to facilitate separation and reuse. In these studies we report on the selection of appropriate enzyme-carrier preparation for hydrolysis reaction in aqueous and biphasic systems and transesterification in organic solvent. For this Candida rugosa lipase was bound by adsorption or covalent attachment onto various carriers to give 24 preparations. Selection of proper preparation was based on reactivity, thermal stability (4 h at 60 o C), possibility of drying and operational stability in 17-23 successive batch processes of 4-nitrophenyl palmitate hydrolysis in water. Activity of preparations varied from 20 to 5100 U•mL -1 but the most stable preparations were those of moderate activity: bound by adsorption or covalent attachment to NH 2 -Kieselgel or acrylic carrier (retained activity over 90%). Selected preparations were used for hydrolysis of ethyl (1-butyryloxyethyl)-phenylphosphinate in biphasic system, and, after drying, in ethyl (1-hydroxyethyl)-phenylphosphinate transesterification. In this study operational stability was the principal criterion of selection. In water system, lipase covalently bound to NH 2 -Kieselgel was the best -preserved 50% of initial activity in consecutive batch processes. In biphasic system and lipase covalently bound to acrylic and NH 2 -Kieselgel the values were 90 or 77%, respectively, whereas in organic solvent, when lipase was immobilized on NH 2 -Kieselgel by adsorption, it was 50%. Thus, NH 2 -Kieselgel appears to be an universal matrix for investigated lipase immobilization and can be used in all reaction systems

Screening of lipase carriers for reactions in water, biphasic and pure organic solvent systems

In bioprocesses lipases are typically used in immobilized form, irrespective of type of reaction systems, to ensure an even distribution of catalysts in water restricted media and/or to facilitate separation and reuse. In these studies we report on the selection of appropriate enzyme-carrier preparation for hydrolysis reaction in aqueous and biphasic systems and transesterification in organic solvent. For this Candida rugosa lipase was bound by adsorption or covalent attachment onto various carriers to give 24 preparations. Selection of proper preparation was based on reactivity, thermal stability (4 h at 60°C), possibility of drying and operational stability in 17-23 successive batch processes of 4-nitrophenyl palmitate hydrolysis in water. Activity of preparations varied from 20 to 5100 U∙mL-1 but the most stable preparations were those of moderate activity: bound by adsorption or covalent attachment to NH2-Kieselgel or acrylic carrier (retained activity over 90%). Selected preparations were used for hydrolysis of ethyl (1-butyryloxyethyl)-phenylphosphinate in biphasic system, and, after drying, in ethyl (1-hydroxyethyl)-phenyl-phosphinate transesterification. In this study operational stability was the principal criterion of selection. In water system, lipase covalently bound to NH2-Kieselgel was the best - preserved 50% of initial activity in consecutive batch processes. In biphasic system and lipase covalently bound to acrylic and NH2-Kieselgel the values were 90 or 77%, respectively, whereas in organic solvent, when lipase was immobilized on NH2-Kieselgel by adsorption, it was 50%. Thus, NH2-Kieselgel appears to be an universal matrix for investigated lipase immobilization and can be used in all reaction systems

Stability of Tyrosinase in Presence of Reaction Mixture Components with Borate Buffer.

<p>Relative activity of native (black) and immobilized enzyme (gray) after 1 h incubation at 30°C in 0.5 M borate buffer, pH 9 (Control) containing 1 mM L-tyrosine (L-tyr), 1 mM L-DOPA, 2 mM ascorbic acid (AH₂), or 6.7 mM hydroxylamine (HA).</p

Effective L-Tyrosine Hydroxylation by Native and Immobilized Tyrosinase

<div><p>Hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA) by immobilized tyrosinase in the presence of ascorbic acid (AH₂), which reduces DOPA-quinone to L-DOPA, is characterized by low reaction yields that are mainly caused by the suicide inactivation of tyrosinase by L-DOPA and AH₂. The main aim of this work was to compare processes with native and immobilized tyrosinase to identify the conditions that limit suicide inactivation and produce substrate conversions to L-DOPA of above 50% using HPLC analysis. It was shown that immobilized tyrosinase does not suffer from partitioning and diffusion effects, allowing a direct comparison of the reactions performed with both forms of the enzyme. In typical processes, additional aeration was applied and boron ions to produce the L-DOPA and AH₂ complex and hydroxylamine to close the cycle of enzyme active center transformations. It was shown that the commonly used pH 9 buffer increased enzyme stability, with concomitant reduced reactivity of 76%, and that under these conditions, the maximal substrate conversion was approximately 25 (native) to 30% (immobilized enzyme). To increase reaction yield, the pH of the reaction mixture was reduced to 8 and 7, producing L-DOPA yields of approximately 95% (native enzyme) and 70% (immobilized). A three-fold increase in the bound enzyme load achieved 95% conversion in two successive runs, but in the third one, tyrosinase lost its activity due to strong suicide inactivation caused by L-DOPA processing. In this case, the cost of the immobilized enzyme preparation is not overcome by its reuse over time, and native tyrosinase may be more economically feasible for a single use in L-DOPA production. The practical importance of the obtained results is that highly efficient hydroxylation of monophenols by tyrosinase can be obtained by selecting the proper reaction pH and is a compromise between complexation and enzyme reactivity.</p></div

Selected Parameters of Processes of 1 mM L-tyrosine Hydroxylation Using Native or Immobilized Tyrosinase in Reaction Systems with Borate Buffer (BB; 0.5 M), and/or Ascorbic Acid (AH₂; 2 mM), and/or Hydroxylamine (HA; 6.7 mM).

<p>Selected Parameters of Processes of 1 mM L-tyrosine Hydroxylation Using Native or Immobilized Tyrosinase in Reaction Systems with Borate Buffer (BB; 0.5 M), and/or Ascorbic Acid (AH₂; 2 mM), and/or Hydroxylamine (HA; 6.7 mM).</p

Selected Parameters of Processes of 1 mM L-tyrosine Hydroxylation Using Native or Immobilized Tyrosinase in Phosphate Buffer (0.1 M, pH 7) in the Presence of Ascorbic Acid (AH₂; 2 mM).

<p>Selected Parameters of Processes of 1 mM L-tyrosine Hydroxylation Using Native or Immobilized Tyrosinase in Phosphate Buffer (0.1 M, pH 7) in the Presence of Ascorbic Acid (AH₂; 2 mM).</p

Novel method for synthesis of silver nanoparticles and their application on wool

In this study, a new method for the synthesis of silver nanoparticles (AgNPs) suitable to impart antibacterial properties of wool fabric is proposed. AgNPs were synthesized by a biochemical reduction method. An aqueous solution of extracted dye from Pomegranate peel was used as a reducing agent for the synthesis of AgNPs from silver nitrate. The ratio of dye to silver nitrate concentration (R-Dye/Ag = [Dye]I[AgNO3]) is the influencing factor in the synthesis of silver nanoparticles. The nanoparticles formation was followed by UV/Vis absorption spectroscopy. The size and shape of AgNPs were studied by transmission electron microscopy (TEM). The size distribution and Zetapotential of nanoparticles were evaluated using diffraction light scattering (DLS) measurements. The antibacterial potential of biosynthesized silver nanoparticles against Escherichia coli (E. coli) was examined qualitatively and quantitatively. Kinetic analysis of the bacteria reduction using AgNPs synthesized in different way was performed. AgNPs were applied on wool fabrics by exhaustion. The changes in surface morphology of wool fibers after AgNPs loading were studied using scanning electron microscopy (SEM). The amounts of silver deposited on wool fabrics at different pH and temperature were compared applying energy-dispersive X-ray spectroscopy (EDX). AgNPs loaded fabrics showed excellent antibacterial efficiency even after five washing cycles. To investigate the nature of interaction and bonding between the AgNPs and the wool substrate XPS measurements were performed. (C) 2015 Elsevier B.V. All rights reserved

Simplified Schematic Representation of L-tyrosine Hydroxylation by Tyrosinase.

<p>Reactions without ascorbic acid—black arrows and a reaction system with ascorbic acid (AH₂) supplementation—gray arrow. More detailed information about the actions of tyrosinase on L-tyrosine and L-DOPA is summarized in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164213#pone.0164213.ref002" target="_blank">2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164213#pone.0164213.ref004" target="_blank">4</a>].</p