Jolanta Liesiene

Biomimetic Mineralization on a Macroporous Cellulose-Based Matrix for Bone Regeneration

The aim of this study is to investigate the biomimetic mineralization on a cellulose-based porous matrix with an improved biological profile. The cellulose matrix was precalcified using three methods: (i) cellulose samples were treated with a solution of calcium chloride and diammonium hydrogen phosphate; (ii) the carboxymethylated cellulose matrix was stored in a saturated calcium hydroxide solution; (iii) the cellulose matrix was mixed with a calcium silicate solution in order to introduce silanol groups and to combine them with calcium ions. All the methods resulted in a mineralization of the cellulose surfaces after immersion in a simulated body fluid solution. Over a period of 14 days, the matrix was completely covered with hydroxyapatite crystals. Hydroxyapatite formation depended on functional groups on the matrix surface as well as on the precalcification method. The largest hydroxyapatite crystals were obtained on the carboxymethylated cellulose matrix treated with calcium hydroxide solution. The porous cellulose matrix was not cytotoxic, allowing the adhesion and proliferation of human osteoblastic cells. Comparatively, improved cell adhesion and growth rate were achieved on the mineralized cellulose matrices.

Synthesis and properties of acrylic carriers from acrylonitrile, vinyl acetate and divinylbenzene terpolymers

Abstract Two series of carriers based on acrylonitrile (AN)-vinyl acetate (VA)-divinylbenzene (DVB) terpolymers were obtained by aminolysis of nitrile groups to N-alkylamino ones. In the first series, the terpolymers differed in the content of VA units (7.5, 10, 20, and 30 mol.%). The modifying diluents used in the polymerization stage were: hexadecane (HD) and toluene (T). In the second series, the modifying diluents were: cyclohexanol (C), octanol (O), decanol (D), 2-ethylhexanol (E), and benzyl alcohol (B). The content of VA units was constant (20 mol.%). The IR spectra revealed that the aminolysis of nitrile groups of the terpolymers to N-ethylamino ones was accompanied by hydrolysis of the ester groups. The carriers with hydroxy groups, however, did not swell more than those obtained from AN-DVB copolymers. The concentration of amino groups was ∼ 1.8 mmol/g and the porosity was in the range 0.1–0.5, depending on the kind of diluent used in polymerization. The structural parameters of the carriers were characterized by the inverse GPC method with dextran solutions. The pore size (mean pore diameter) and its distribution width decreased with increasing VA unit content for the series HDT-7.5-HDT-30. In the second series, the porosity characteristics depended on the molecular shape of the thermodynamically bad diluent used. Acylase immobilized on those carriers had smaller activity than that immobilized on previously described carriers, whereas for glucoamylase and peroxydase the results of immobilization were very promising.

Interaction of cellulose-based cationic polyelectrolytes with mucin.

Mucoadhesivity of water-soluble polymers is an important factor, when testing their suitability for controlled drug delivery systems. For this purpose, the interaction of new cationic cellulose polyelectrolytes with lyophilized mucin was investigated by means of turbidimetric titration, microscopy and measurement of zeta potential and particle size changes in the system. Results show that the cellulose derivatives interact with mucin. This interaction became stronger if cellulose macromolecules contained positively charged groups and an electrostatic interaction with the negatively charged mucin particles occurred. Under certain conditions flocculation of mucin particles by the cellulose polyelectrolyte was observed.

Effective L-Tyrosine Hydroxylation by Native and Immobilized Tyrosinase

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.

Flocculation of Bacillus amyloliquefaciens H Disintegrates with Cationized Starch and Aminated Hydroxyethylcellulose

The ability of cationized hydroxyethylated starch (CHES) and aminated hydroxyethylcellulose (DEAE-HEC) to flocculate cell disintegrate of Bacillus amyloliquefaciens H (BamHI) was investigated. The efficiency of flocculation was compared to that of synthetic polymer polyethyleneimine (PEI) and natural polysaccharide chitosan. The influence of salt concentration and biomass concentration on flocculation efficiency was investigated. It was found that the efficiency of flocculation with CHES and DEAE-HEC was similar to that of PEI but better compared to chitosan. Recovery of total soluble proteins at higher than 0.3% concentration of flocculant decreased by more than 18.8% and 42.3% compared to PEI and chitosan, respectively. The yield of BamHI restriction endonuclease activity with all flocculants was similar except for chitosan where 13.1% lower yield was obtained. Meanwhile, efficiency of flocculation with CHES and DEAE-HEC depends drastically on the salt concentration, that is, flocculation diminishes if NaCl concentrations higher than 0.2 M (for CHES) or 0.1 M (for DEAE-HEC) are used. The results have shown that CHES and DEAE-HEC are promising flocculants of cell disintegrates if higher yield of protein is of great concern.

Influence of Surface-Active Compounds on Starch Dispersion in Water. Part I. Starch Pasting

The influence of native lipids and additives of surface-active compounds on starch paste rheology was investigated. The aim of the study was to gain better understanding of mechanisms involved in starch gelatinization and how these structure changes of granules later affect rheological properties of pastes and gels. Starches from three main sources—potato, maize, and wheat—were tested; sodium dodecylsulfate, oleate, and benzalkonium chloride were employed as additives. Starch pasting was examined by a rheometer to get a viscosity profile, also pastes were analyzed by differential scanning calorimetry, for particle size using a light scattering technique. Results revealed that there was a competition between native lipids and added surfactants for amylose complexation. Complexes formed during gelatinization were strongly affecting granule swelling and dissolution of starch polymers, and viscosity of pastes was mainly dependent on the particle size of a disperse phase in the paste. Addition of strong ionic surfactants to cereal starches resulted in smaller granular remnants and, therefore, decreased viscosity, while the weak anionic surfactant promoted an increase in the particle size and paste viscosity for both cereal and tuber starches. The mechanism of the effect of surfactants on the particle size in pastes is discussed.

ANION EXCHANGE CHROMATOGRAPHY OF PROTEINS. EFFECT OF CHARGED GROUPS OF THE STATIONARY PHASE ON PROTEIN BINDING

The purpose of this work was to study how the basicity and density of charged groups of anion exchangers affect protein binding strength and chromatographic performance at different sample loading. For this study, several cellulose-based anion exchangers with different ligand basicity and quantity were synthesized. The binding strength of protein by the ion exchangers, and the chromatographic performance were evaluated. Investigations showed that a significant part of the protein adsorbed in the high loading mode may be eluted with 0.05–0.1 M NaCl in Tris-HCl buffer pH 7.5. This indicates very weak binding. It may be the result of multi-layer binding of protein molecules to that already adsorbed on the surface of stationary phase. The amount of weakly bound protein decreases with decreasing of sample loading. Results of gradient elution of BSA adsorbed at low loading mode showed that the binding strength of protein is dependent on the basicity of ionic groups of the stationary phase. A tendency towards improved chromatographic performance was observed with increasing density of charged groups on the surface of the stationary phase.

Influence of Surface Active Compounds on Starch Dispersion in Water. Part II. Short-Term Retrogradation

The effect of native lipids and additives of surface active compounds on starch short-term retrogradation and formed gel rheology was investigated. The main aim of the study was to correlate the findings of pasting analysis with the phenomena observed during starch retrogradation and gel formation as affected by the surface active compounds. Three commercially most important starches (potato, maize, and wheat) were tested; sodium dodecyl sulfate, oleate, and benzalkonium chloride were employed as the additives. The study revealed that the rheology of retrograded starch pastes mainly depended on the size of granule residues, amounts of amylose available for gel matrix formation, and a surfactant ability to form inclusion complexes with amylose. The latter factor was evidenced by overlapping of the inclusion complex formation temperatures with the stage of a rapid increase in G′ module of starch gels on cooling. Also the competition of native and added surfactants for the complex formation with amylose was evidenced by shifts in the temperature of complex formation. Starch dispersion in water could be regulated through the control of all three factors by adding a proper surfactant to dispersion of starch, depending on its botanical origin. GRAPHICAL ABSTRACT

COMPARATIVE STUDY OF MACROPOROUS SILICA- AND CELLULOSE-BASED SORBENTS FOR LECTIN AFFINITY CHROMATOGRAPHY

□ Preparative lectin affinity chromatography is widely used as a first step for the isolation and purification of glycoproteins. In this work lectin affinity adsorbents for preparative processes were prepared on different macroporous matrices: silica coated with polyvinyl alcohol or its cationic derivative, and cellulose. Activation of the matrices with carbonyldiimidazole and immobilization of Concanavalin A (ConA) was studied. The chromatographic performance of prepared adsorbents was evaluated by means of glucose oxidase (GOD) adsorption isotherms and breakthrough curves. It was found that the adsorption isotherms include two steps. It was supposed that the second step might be a result of GOD dimmer adsorption from GOD solutions of a high concentration. The dissociation constant of the affinity pair was found in the magnitude of 10−6 to 10−7 mol L−1. Breakthrough curves showed that the sorption process on a soft cellulose-based adsorbent was highly influenced by the diffusion. Thus, the flow rate applicable for cellulose-based adsorbents should be lower than that for silica ones. In both static and dynamic GOD adsorption conditions a higher sorption capacity was obtained on the cellulose-based adsorbent, although kinetic parameters were better on silica-based ones. Moreover, despite the surface coating with hydrophilic polymers, silica adsorbents possessed high non-specific sorption of the protein.