Influence of the composition and preparation method on the morphology and swelling behavior of alginate–chitosan hydrogels

In this study, a 2⁴ factorial experimental design was employed in order to evaluate the influence of the reaction conditions and preparation method on alginate–chitosan hydrogel properties. Alginate content, pH, chitosan molecular weight and the hydrogel preparation method were the independent variables and the reaction yield, particle size, swelling degree and point of zero surface charge were the dependent variables. The results showed that hydrogels were spherical with an average diameter of 5.0 ± 2.0 μm. Reaction yield varied according to the parameters, and chitosan molecular weight showed the greatest influence. Furthermore, the swelling degree and point of zero surface charge showed a linear dependence on the alginate content. In this regard, the study showed that hydrogels with a specific charge and swelling degree can be obtained by controlling the alginate content using the equation here provided to give an enhanced and site-specific controlled drug release.     

Immobilization of fructosyltransferase by chitosan and alginate for efficient production of fructooligosaccharides

The effective system of reusing mycelial fructosyltransferase (FTase) immobilized with two polymers, chitosan and alginate were evaluated for continuous production of fructooligosaccharides (FOS). The alginate beads were successfully developed by maintaining spherical conformation of using 0.3% (w/v) sodium alginate with 0.1% (w/v) of CaCl₂ solution for highest transfructosylating activity. The characteristics of free and immobilized FTase were investigated and results showed that optimum pH and temperature of FTase activity were altered by immobilized materials. A successive production of FOS by FTase entrapped alginate beads was observed at an average of 62.96% (w/w) up to 7 days without much losing its activity. The data revealed by HPLC analysis culminate 67.75% (w/w) of FOS formation by FTase entrapped alginate beads and 42.79% (w/w) by chitosan beads in 36 h of enzyme substrate reaction.     

Swelling behaviour of alginate–chitosan microcapsules prepared by external gelation or internal gelation technology

Swelling behaviour is one of the important properties for microcapsules made by hydrogels, which always affects the diffusion and release of drugs when the microcapsules are applied in drug delivery systems. In this paper, alginate–chitosan microcapsules were prepared by different technologies called external or internal gelation process respectively. With the volume swelling degree (S_w) as an index, the effect of properties of chitosan on the swelling behaviour of both microcapsules was investigated. It was demonstrated that the microcapsules with low molecular weight and high concentration of chitosan gave rise to low S_w. Considering the need of maintaining drug activity and drug loading, neutral pH and short gelation time were favorable. It was also noticed that S_w of internal gelation microcapsules was lower than that of external gelation microcapsules, which was interpreted by the structure analysis of internal or external gelation Ca–alginate beads with the aid of confocal laser scanning microscope.     

Locomotion and adhesion of neutrophil granulocytes : Effects of albumin, fibrinogen and gamma globulins studied by reflection contrast microscopy

Proteins as well as materials of low molecular weight have marked effects on the rate of locomotion, adhesion and cell shape of human neutrophil granulocytes in vitro. Plasma protein preparations differ qualitatively with respect to their chemokinetic activity. Human serum albumin (HSA), fibrinogen and acid-treated gamma globulin without polymers have a positive chemokinetic effect on neutrophils suspended in Gey's solution. Standard gamma globulin (SGG) or acid-treated gamma globulin with polymers have marked negative chemokinetic activity. Three different mechanisms are presumably responsible for the low rate of locomotion observed in Gey's solution alone, Gey's solution containing acid-treated gamma globulin with polymers or SGG, respectively: (a) too firm adhesion to the substratum; (b) lack of adhesion to the substratum; and (c) impaired capacity to perform shape changes. The relationship between attachment of cells to the substratum and the rate of neutrophil locomotion has been investigated. It appears that the pattern of adhesion rather than cell attachment as measured by the proportion of neutrophils adhering to the substratum is a meaningful correlate to locomotion. Two different patterns of adhesion can be distinguished by means of reflection-contrast microscopy: (a) the pattern characterized by uniform grey areas is compatible with efficient locomotion; (b) a pattern characterized by large black areas at the cell periphery. It is associated with neutrophils in Gey's solution which fail to displace themselves efficiently. This suggests that reflection-contrast microscopy may be helpful in distinguishing contacts allowing locomotion to occur from contacts impeding neutrophil locomotion.     

Microstructural characterization of chitosan and alginate films by microscopy techniques and texture image analysis

The aim of this work is to characterize the microstructure of chitosan and alginate edible films by microscopy techniques and texture image analysis. Edible films were obtained by solution casting and solvent evaporation. The microscopy techniques used in this work were: light, environmental scanning electron and atomic force microscopy. Textural features and fractal dimension were extracted from the images. Entropy and fractal dimension were more useful to evaluate the complexity and roughness of films. The highest values of entropy and fractal dimension corresponded to alginate/chitosan, followed of alginate and chitosan films. An entropy/fractal dimension ratio, proposed here, was useful to characterize the degree of image complexity and roughness of edible films at different magnifications. It was possible to postulate that microscopy techniques combined with texture image analysis are efficient tools to quantitatively evaluate the surface morphology of edible films made of chitosan and alginate.     

Encapsulation of an Agrobacterium radiobacter extract containing d-hydantoinase and d-carbamoylase activities into alginate–chitosan polyelectrolyte complexes: Preparation of the biocatalyst

Alginate–chitosan polyelectrolyte complexes (PECs) have been used for the first time as a suitable matrix for coimmobilisation of enzymes to reproduce a multistep enzymatic route for production of d-amino acids. Encapsulation of a crude cell extract from Agrobacterium radiobacter containing d-hydantoinase and d-carbamoylase activities into the PECs with negligible leakage from the formed capsules was accomplished. All results in this study indicate that the preparation of the biocatalyst (preparation method and chitosan characteristics) play a key role in the biocatalyst's properties. The most suitable biocatalysts were prepared using a chitosan with a medium molecular weight (600 kDa) and a degree of deacetylation of 0.9. For all of the preparation conditions under study, an encapsulation yield of around 60% was achieved and the enzymatic activity yields ranged from 30 to 80% for d-hydantoinase activity and from 40 to 128% for d-carbamoylase activity relative to the activities of the soluble extract. All of the biocatalysts were able to hydrolyze l,d-hydroxyphenylhydantoin into p-hydroxyphenylglycine with yields ranging from 30 to 80%.     

Calcium alginate–starch hybrid support for both surface immobilization and entrapment of bitter gourd (Momordica charantia) peroxidase

Calcium alginate–starch hybrid gel was employed as an enzyme carrier both for surface immobilization and entrapment of bitter gourd peroxidase. Entrapped crosslinked concanavalin A–bitter gourd peroxidase retained 52% of the initial activity while surface immobilized and glutaraldehyde crosslinked enzyme showed 63% activity. A comparative stability of both forms of immobilized bitter gourd peroxidase was investigated against pH, temperature and chaotropic agent; like urea, heavy metals, water-miscible organic solvents, detergent and inhibitors. Entrapped peroxidase was significantly more stable as compared to surface immobilized form of enzyme. The pH and temperature-optima for both immobilized preparations were the same as for soluble bitter gourd peroxidase. Entrapped crosslinked concanavalin A–bitter gourd peroxidase showed 75% of the initial activity while the surface immobilized and crosslinked bitter gourd peroxidase retained 69% of the original activity after its seventh repeated use.     

Insulin-loaded alginate microspheres for oral delivery – Effect of polysaccharide reinforcement on physicochemical properties and release profile

Oral administration of insulin requires protein protection from degradation in the gastric environment and its absorption improvement in the intestinal tract. To achieve this objective several types of microspheres composed of alginate, chitosan and dextran sulphate have been prepared by ionotropic gelation. Parameters such as the mean particle size, swelling behaviour, insulin encapsulation efficiency, loading capacity and release profiles in simulated gastric and intestinal fluids have been compared for the systems developed. In this study, attempts have been made to increase insulin protection and to improve its release from microspheres by reinforcing the alginate matrix with chitosan and/or dextran sulphate. Dextran sulphate was able to avoid insulin release at pH 1.2, protecting the protein from the acidic environment and reducing the total insulin released at pH 6.8. This effect was explained by an interaction between the permanent negatively charged groups of dextran sulphate and insulin molecules.     

Synthesis and bioactivities of poly(ethylene glycol)–chitosan hybrids

Poly(ethylene glycol)–chitosan hybrids of various molecular weights having different degree of substitution were synthesized, by reductive N-alkylation of chitosan with poly(ethylene glycol) aldehyde, to study their bioactivities. The influence of these chitosan derivatives on the reactive oxygen species generation from canine polymorphonuclear leukocyte cells was investigated in vitro by chemiluminescence response. Reactive oxygen species generation by the influence of poly(ethylene glycol)–chitosan hybrids was decreased with the increase of degree of substitution. The reduction of interaction of poly(ethylene glycol)–chitosan hybrids with polymorphonuclear leukocyte cells might be caused by the decrease of amino group in chitosan main chain and increase of the steric hindrance by poly(ethylene glycol) chain. The influence of the poly(ethylene glycol)–chitosan hybrids on complement component C3 activation was investigated by single radial immunodiffusion method. Influence on complement component C3 activation by poly(ethylene glycol)–chitosan hybrids was almost same as chitosan.     

Changes in the Mark–Houwink hydrodynamic volume of chitosan molecules in solutions of different organic acids, at different temperatures and ionic strengths

The objective of this study is to explore the cause(s) of changes in the hydrodynamic volume of chitosan molecules in solutions of different organic acids, at different temperatures and ionic strengths. Change in intrinsic viscosity is used as the parameter to elucidate the causes of changes in the hydrodynamic volume of chitosan molecules in these solutions. Results show that the intrinsic viscosity of chitosan decreases in acetic acid or in malic acid over storage time. These decreases are more pronounced in acetic acid solution than in malic acid solution, more significant in higher temperature than in lower temperature solutions, and greater in solutions without NaCl than in solutions containing higher NaCl. The decrease in intrinsic viscosity can perhaps be attributed to the compounded effects of compaction of the chitosan molecules and/or acidic degradation during storage.     

Binding and biological properties of lipopolysaccharide Proteus vulgaris O25 (48/57)–chitosan complexes

In this study the negatively charged Proteus vulgaris O25 LPS was chosen for studying interaction with polycationic chitosan. The complex formation of LPS with chitosan was demonstrated using gradient centrifugation and laser interferometry method. The presented results have shown that laser interferometry method is sensitive enough for LPS–chitosan interaction studies. The changing in the ultra structure of LPS during binding with chitosan was observed by electronic microscope. The interaction of P. vulgaris O25 LPS with chitosan was shown to modulate significantly the biological activities of LPS. The toxicity of P. vulgaris O25 LPS decreased 10-fold after forming complexes with chitosan at injection to mice in the similar concentration of endotoxin. The complex LPS–chitosan was less effective than LPS alone in Limulus amabocyte lysate assay. Induction of TNF biosynthesis by LPS–chitosan complex was found to be 65% lower than that by parent LPS at concentration of 100 ng/ml.     

Cross-linking properties of alginate gels determined by using advanced NMR imaging and Cu<Superscript>2+</Superscript> as contrast agent

The entrapment of enzymes, drugs, cells or tissue fragments in alginates cross-linked with Ca²⁺ or Ba²⁺ has great potential in basic research, biotechnology and medicine. The swelling properties and, in turn, the mechanical stability are key factors in designing an optimally cross-linked hydrogel matrix. These parameters depend critically on the cross-linking process and seemingly minor modifications in manufacture have a large impact. Thus, sensitive and non-invasive tools are required to determine the spatial homogeneity and efficacy of the cross-linking process. Here, we show for alginate microcapsules (between 400 µm and 600 µm in diameter) that advanced ¹H NMR imaging, along with paramagnetic Cu²⁺ as contrast agent, can be used to validate the cross-linking process. Two- and three-dimensional images and maps of the spin-lattice relaxation time T₁ of Ba²⁺ cross-linked microcapsules exposed to external Cu²⁺ yielded qualitative as well as quantitative information about the accumulation of Cu²⁺ within and removal from microcapsules upon washing with Cu²⁺ free saline solution. The use of Cu²⁺ (having a slightly higher affinity constant to alginate than Ba²⁺) for gelling gave a complementary insight into the spatial homogeneity of the cross-linking process together with information about the mechanical stability of the microcapsules. The potential of this technique was demonstrated for alginates extracted from two different algal sources and cross-linked either externally by the conventional air-jet dropping method or internally by the "crystal gun" method.     

Evolution of gold nanoparticle clusters in living cells studied by sectional dark‐field optical microscopy and chromatic analysis

The evolution of gold nanoparticle (Au NP) clusters in living cells are studied by using sectional dark‐field optical microscopy and chromatic analysis approach. During endocytosis, Au NP clusters undergo fantastic color changes, from green to yellow‐orange due to the plasmonic coupling effect. Analysis of brightness/hue values of the dark‐field images helps estimate the numbers of Au NPs in the clusters. The Au NP clusters were further categorized into four groups within the endocytosis. As the results, the late endosomes had increased number of large Au NP clusters with time, while clustered numbers in secondary and tertiary groups were first increased and then decreased due to the fusion and fission of the endocytic vesicles. The time constants and cluster numbers for different groups are fitted by using an integrated rate equation, and show a positive correlation with the size of the Au NP cluster. The efficiency of Au NP uptake is only about 50% for normal cells, while 75% for cancer cells. Compared to normal cells, cancer cells show a larger number in uptake, while faster rate in removal. The propose method helps the kinetic study of endocytosed nanoparticles in physiological conditions.

     

Teliospores of smut fungi teliospore connections, appendages, and germ pores studied by electron microscopy; phylogenetic discussion of characteristics of teliospores

Summary Special features of teliospores in smut fungi are described, including teliospore connections, appendages, and germ pores. Balls of teliospores in species of many different genera cohere by remnants of hyphal walls, sheaths, and sometimes interlocking ornamentation. Teliospores are connected in pairs in species ofMycosyrinx andGeminago by special local structures. Appendages can be formed locally by persistent material from the sheath (Cintractia, Anthracoidea, Sphacelotheca), thickened parts of the spore wall (e.g.,Georgefischeria, Jamesdicksonia, Rhamphospora, Tolyposporella), or persistent walls of sporogenous hyphae (Rhamphospora, genera of the Tilletia relationship). Species ofGeorgefischeria, Jamesdicksonia, andTolyposporella have teliospore walls composed of more than three layers of different electron density. Germ areas corresponding to thinner parts of the spore wall are known, e.g., for species ofAnthracoidea, Cintractia, andUstilago infecting members of the family Poaceae, while distinct germ pores, one per teliospore, are found in some species ofThecaphora, Tolyposporium, andSporisorium. Teliospores ofMycosyrinx cissi have a germination ring. Characteristics of teliospores are used to discuss the phylogeny of smut fungi. A phylogenetic tree in accordance with teliospore characteristics is compared to those obtained from ultrastructural characteristics of host-parasite interaction, of septal pores, and from sequence data. Aspects of teliospore development help to define taxa at a high systematic level (Entorrhizales, Ustilaginales, Tilletiales/Entylomatales, Microbotryaceae), while details of ornamentation ontogeny delimit groups of genera (e.g., genera related toUstilago on members of the Poaceae andSporisorium, Cintractia andAnthracoidea, Tilletia) or single genera (e.g.,Melanopsichium, Dermatosorus, Mycosyrinx, Doassinga, Rhamphospora). Types of ornamentation (warty, reticulate), middle layers, teliospore balls, and germ pores evolved repeatedly by convergence. The smut teliospore itself probably evolved independently at least twice, or perhaps three (or more) times, in the Microbotryales, in the Entorrhizales, and in a common ancestor of the remainder of the Ustilaginomycetes.Part 166 in the series Studies in Heterobasidiomycetes from the Botanical Institute, University of Tübingen.     

Quantitative analysis of shape and volume changes in activated thrombocytes in real time by single‐shot spatial light modulator‐based differential interference contrast imaging

We suggest to use a combination of optical tweezers and single‐image quantitative differential interference contrast (DIC) emulated by a spatial light modulator (SLM) to study physiological shape changes in thrombocytes after activation and demonstrate the effectiveness of this system for the given task. A specially designed phase mask displayed at the SLM enables quantitative phase calculation from only a single recording. The optical tweezers stabilize trapped thrombocytes for long‐time monitoring of changes in the optical thickness profile of thrombocytes during activation by adenosine diphosphate (ADP). (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Conformational behavior of the HAV-VP3(110–121) peptidic sequence and synthetic analogs in membrane environments studied by CD and computational methods

The present study was undertaken to examine the structural features that may be important to explain the immunogenicity of the (110–121) peptide sequence (FWRGDLVFDFQV) of VP3 capsid protein of hepatitis A virus. A conformational analysis of the preferred conformations by CD and molecular mechanics was carried out. Present results suggest that the interaction with liposomes as biomembrane model induces and stabilizes the amphipathic β-structure of the peptide. To study the contribution of amino acid replacements at the RGD tripeptide as well as the influence of the peptide chain length on peptide conformation, solid-phase peptide synthesis of several peptide analogs was carried out and the peptide conformation was studied using CD spectroscopy. The results show that the RGD sequence is necessary to induce the β-structure in the presence of liposomes. © 1998 John Wiley & Sons, Inc. Biopoly 45: 479–492, 1998     

Finish line plant–insect interactions mediated by insect feeding mode and plant interference: a case study of Brassica interactions with diamondback moth and turnip aphid

There are gaps in our understanding of plant responses under different insect phytophagy modes and their subsequent effects on the insect herbivores’ performance at late season. Here we compared different types of insect feeding by an aphid, Lipaphis erysimi, and a lepidopteran, Plutella xylostella, and how this affected defensive metabolites in leaves of 2 Brassica species when plants gain maturity. Thiocyanate concentrations after P. xylostella and L. erysimi feeding activities were the same. Total phenolics was higher after the phloem feeder feeding than the folivore activity. The plants compensatory responses (i.e., tolerance) to L. erysimi feeding was significantly higher than the responses to P. xylostella. This study showed that L. erysimi had higher carbon than P. xylostella whereas nitrogen in P. xylostella was 1.42 times that in L. erysimi. Population size of the phloem feeder was not affected by plant species or insect coexistence. However, there was no correlation between plant defensive metabolites and both insects’ population size and biomass. This suggests that plant root biomass and tolerance index after different insect herbivory modes are not necessarily unidirectional. Importantly, the interaction between the folivore and the phloem feeder insects is asymmetric and the phloem feeder might be a trickier problem for plants than the folivore. Moreover, as both plants’ common and special defenses decreased under interspecific interference, we suggest that specialist insect herbivores can be more challenged in ecosystems in which plants are not involved in interspecific interference.