Fast and Sustained Axonal Growth by BDNF Released from Chitosan Microspheres.

Brain-derived neurotrophic factor (BDNF) regulates dendritic branching and dendritic spine morphology, as well as synaptic plasticity and long-term potentiation. Consequently, BDNF deficiency has been associated with some neurological disorders such as Alzheimer's, Parkinson's or Huntington's diseases. In contrast, elevated BDNF levels correlate with recovery after traumatic central nervous system (CNS) injuries. The utility of BDNF as a therapeutic agent is limited by its short half-life in a pathological microenvironment and its low efficacy caused by unwanted consumption of non-neuronal cells or inappropriate dosing. Here, we tested the activity of chitosan microsphere-encapsulated BDNF to prevent clearance and prolong the efficacy of this neurotrophin. Neuritic growth activity of BDNF release from chitosan microspheres was observed in the PC12 rat pheochromocytoma cell line, which is dependent on neurotrophins to differentiate via the neurotrophin receptor (NTR). We obtained a rapid and sustained increase in neuritic out-growth of cells treated with BDNF-loaded chitosan microspheres over control cells (p < 0.001). The average of neuritic out-growth velocity was three times higher in the BDNF-loaded chitosan microspheres than in the free BDNF. We conclude that the slow release of BDNF from chitosan microspheres enhances signaling through NTR and promotes axonal growth in neurons, which could constitute an important therapeutic agent in neurodegenerative diseases and CNS lesions.

Influence of Preparation Methods of Chitooligosaccharides on Their Physicochemical Properties and Their Anti-Inflammatory Effects in Mice and in RAW264.7 Macrophages.

The methods to obtain chitooligosaccharides are tightly related to the physicochemical properties of the end products. Knowledge of these physicochemical characteristics is crucial to describing the biological functions of chitooligosaccharides. Chitooligosaccharides were prepared either in a single-step enzymatic hydrolysis using chitosanase, or in a two-step chemical-enzymatic hydrolysis. The hydrolyzed products obtained in the single-step preparation were composed mainly of 42% fully deacetylated oligomers plus 54% monoacetylated oligomers, and they attenuated the inflammation in lipopolysaccharide-induced mice and in RAW264.7 macrophages. However, chitooligosaccharides from the two-step preparation were composed of 50% fully deacetylated oligomers plus 27% monoacetylated oligomers and, conversely, they promoted the inflammatory response in both in vivo and in vitro models. Similar proportions of monoacetylated and deacetylated oligomers is necessary for the mixtures of chitooligosaccharides to achieve anti-inflammatory effects, and it directly depends on the preparation method to which chitosan was submitted.

Chitosan Spray-Dried Microparticles for Controlled Delivery of Venlafaxine Hydrochloride.

Venlafaxine controlled drug delivery systems using different matrixes have been tested to reduce undesirable side effects in the treatment of depression. The legal status of chitosan (Cs) in Pharmacy has dramatically improved after its acceptance as excipient in several Pharmacopeias and, therefore, there is great interest in pharmaceutical formulations based on this polymer. In this paper, chitosan microcapsules cross-linked with sodium tripolyphosphate (TPP) for oral delivery of venlafaxine were formulated using the spray drying technique. The effect of chitosan physico-chemical properties, TPP concentration and TPP/Cs ratio on drug release was evaluated. The microcapsules were characterized in terms of size, zeta potential and morphology. The physical state of the drug was determined by X-ray diffraction (XRD) and the drug release from the microcapsules was studied in simulated gastric and intestinal fluids. The release pattern fitted well to the Peppas-Koersmeyer model with n exponents indicating anomalous transport.

Physical Stability Studies of Semi-Solid Formulations from Natural Compounds Loaded with Chitosan Microspheres.

A chitosan-based hydrophilic system containing an olive leaf extract was designed and its antioxidant capacity was evaluated. Encapsulation of olive leaf extract in chitosan microspheres was carried out by a spray-drying process. The particles obtained with this technique were found to be spherical and had a positive surface charge, which is an indicator of mucoadhesiveness. FTIR and X-ray diffraction results showed that there are not specific interactions of polyphenolic compounds in olive leaf extract with the chitosan matrix. Stability and release studies of chitosan microspheres loaded with olive leaf extract before and after the incorporation into a moisturizer base were performed. The resulting data showed that the developed formulations were stable up to three months. The encapsulation efficiency was around 44% and the release properties of polyphenols from the microspheres were found to be pH dependent. At pH 7.4, polyphenols release was complete after 6 h; whereas the amount of polyphenols released was 40% after the same time at pH 5.5.

Magnetic chitosan beads for covalent immobilization of nucleoside 2'-deoxyribosyltransferase: application in nucleoside analogues synthesis.

Cross-linked magnetic chitosan beads were prepared in presence of epichlorohydrin under alkaline conditions, and subsequently incubated with glutaraldehyde in order to obtain an activated support for covalent attachment of nucleoside 2'-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT). Changing the amount of magnetite (Fe(3)O(4)) and epichlorohydrin (EPI) led to different macroscopic beads to be used as supports for enzyme immobilization, whose morphology and properties were characterized by scanning electron microscopy, spin electron resonance (ESR), and vibrating sample magnetometry (VSM). Once activated with glutaraldehyde, the best support was chosen after evaluation of immobilization yield and product yield in the synthesis of thymidine from 2'-deoxyuridine and thymine. In addition, optimal conditions for highest activity of immobilized LrNDT on magnetic chitosan were determined by response surface methodology (RSM). Immobilized biocatalyst retained 50 % of its maximal activity after 56.3 h at 60 °C, whereas 100 % activity was observed after storage at 40 °C for 144 h. This novel immobilized biocatalyst has been successfully employed in the enzymatic synthesis of 2'-deoxyribonucleoside analogues as well as arabinosyl-nucleosides such as vidarabine (ara-A) and cytarabine (ara-C). Furthermore, this is the first report which describes the enzymatic synthesis of these arabinosyl-nucleosides catalyzed by an immobilized nucleoside 2'-deoxyribosyltransferase. Finally, the attached enzyme to magnetic chitosan beads could be easily recovered and recycled for 30 consecutive batch reactions with negligible loss of catalytic activity in the synthesis of 2,6-diaminopurine-2'-deoxyriboside and 5-trifluorothymidine.

Suitability of a colorimetric method for the selective determination of chitosan in dietary supplements.

A colorimetric method previously described for the determination of chitosan has been evaluated because lack of linearity had been observed at certain concentrations. Calibration curves of varied-characteristic chitosans, recovery studies and chitosan quantification in seven commercial dietary supplements have been performed. Some analysis conditions including the solvent of the samples have been studied and optimised. Different data combinations have been checked in order to select the widest range of concentrations where no serial correlation was found. With the selected conditions the method is linear, reproducible and provides reliable results in the analysis of the chitosan content in capsules. Its selectivity has been proved by the lack of interference with other compounds present in the dietary supplements. But in the case of tablet products, the presence of cellulose and magnesium stearate may produce an underestimation of the chitosan content.

Chitosan derivatives-based films as pH-sensitive drug delivery systems with enhanced antioxidant and antibacterial properties.

The purpose of this study was to develop and characterize chitosan (Ch)-based films incorporated with varying molecular weight (Mw) and acetylation degree (AD) chitosan-depolymerization-products (CDP), to be applied as drug delivery materials. As compared to Ch-film, optical and antioxidant potentials of Ch/CDP-based films were improved, particularly using low Mw and AD-CDP. Whereas, films water resistance, mechanical and antibacterial properties increased as CDP-Mw increased and AD decreased. For the thermal and swelling behaviors, better values were obtained using higher Mw and AD-CDP. Further, to assess their in vitro ciprofloxacin (CFX)-release behavior, loaded-CFX Ch/CDP-based films, crosslinked using glutaraldehyde, were prepared. Expect of elongation at break, crosslinked CFX-loaded films showed increased optical, water resistance, tensile strength and thermal properties, as compared to unloaded films. The CFX-release profiles indicated that a slower and sustained release was observed, particularly when using lower Mw and AD-CDP, and mainly for the crosslinked films during 48 h. These films can release CFX for up to 54% in 6 and 24 h, at pH 1.2 and 7.4, respectively. Through this study, novel biodegradable, swellable and pH-sensitive crosslinked Ch/CDP-based films may be considered as suitable and promising drug delivery systems.

Chitosan-genipin microspheres for the controlled release of drugs: clarithromycin, tramadol and heparin.

The aim of this study was to first evaluate whether the chitosan hydrochloride-genipin crosslinking reaction is influenced by factors such as time, and polymer/genipin concentration, and second, to develop crosslinked drug loaded microspheres to improve the control over drug release. Once the crosslinking process was characterized as a function of the factors mentioned above, drug loaded hydrochloride chitosan microspheres with different degrees of crosslinking were obtained. Microspheres were characterized in terms of size, morphology, drug content, surface charge and capacity to control in vitro drug release. Clarithromycin, tramadol hydrochloride, and low molecular weight heparin (LMWH) were used as model drugs. The obtained particles were spherical, positively charged, with a diameter of 1-10 microm. X-Ray diffraction showed that there was an interaction of genipin and each drug with chitosan in the microspheres. In relation to the release profiles, a higher degree of crosslinking led to more control of drug release in the case of clarithromycin and tramadol. For these drugs, optimal release profiles were obtained for microspheres crosslinked with 1 mM genipin at 50 °C for 5 h and with 5 mM genipin at 50 °C for 5 h, respectively. In LMWH microspheres, the best release profile corresponded to 0.5 mM genipin, 50 °C, 5 h. In conclusion, genipin showed to be eligible as a chemical-crosslinking agent delaying the outflow of drugs from the microspheres. However, more studies in vitro and in vivo must be carried out to determine adequate crosslinking conditions for different drugs.

Controlled size green synthesis of bioactive silver nanoparticles assisted by chitosan and its derivatives and their application in biofilm preparation.

The aim of this work was to explore the effect of various molecular weight (Mw) chitosan depolymerization products (CDP) on the silver nanoparticles (AgNPs) and chitosan/AgNPs blend films production. Produced AgNPs, stable during 30 days in a colloïdal form, were characterized in terms of UV-vis, transmission electron microscopy (TEM), dynamic light scattering (DLS) and fourier transform infrared spectroscopy (FTIR) analyses. AgNPs displayed interesting antibacterial and antioxidant properties that were affected by the physicochemical properties of used chitosans. Interestingly, CDP may be used for the preparation of bioactive and stable AgNPs. Additionally, chitosan/AgNPs blend films were prepared and characterized in terms of physiochemical and biological properties. As compared to the chitosan film, various properties were enhanced in the chitosan/AgNPs blend films, including light barrier, opacity, elongation at break, as well as bioactivities, thus suggesting that films could be used as novel alternative food packaging applications.

Enzymatic production of low-Mw chitosan-derivatives: Characterization and biological activities evaluation.

This study reports the enzymatic depolymerization of chitosan by the chitosanolytic preparation of Bacillus licheniformis GA11. For this purpose, chitosanase production and biochemical characterization of the crude enzyme preparation from GA11 were firstly investigated. The highest chitosanase production was obtained in a culture medium containing 50.0 g/l of a mixture of soluble starch/shrimp shells/crab shells and 5.0 g/l tryptone, after incubation during 5 days, at 30 °C with a pHi of 6.0 and under continuous agitation at 200 rpm. Then, the chitosanolytic preparation, exhibiting maximum activity at 65 °C and pH 5.0, was used to hydrolyze chitosan, leading to various chitosan-depolymerization products (CDP) with different physicochemical characteristics. Finally, the antioxidant and antimicrobial potentials of CDP were evaluated, allowing to conclude that the molecular weight and the acetylation degree highly affect the biological activities of CDP.

Unraveling the Structural Landscape of Chitosan-Based Heparan Sulfate Mimics Binding to Growth Factors: Deciphering Structural Determinants for Optimal Activity.

Chitosan sulfates have demonstrated the ability to mimic heparan sulfate (HS) function. In this context, it is crucial to understand how the specific structural properties of HS domains determine their functionalities and biological activities. In this study, several HS-mimicking chitosans have been prepared to mimic the structure of HS domains that have proved to be functionally significant in cell processes. The results presented herein are in concordance with the hypothesis that sulfated chitosan-growth factor (GF) interactions are controlled by a combination of two effects: the electrostatic interactions and the conformational adaptation of the polysaccharide. Thus, we found that highly charged O-sulfated S-CS and S-DCS polysaccharides with a low degree of contraction interacted more strongly with GFs than N-sulfated N-DCS, with a higher degree of contraction and a low charge. Finally, the evidence gathered suggests that N-DCS would be able to bind to an allosteric zone and is likely to enhance GF signaling activity. This is because the bound protein remains able to bind to its cognate receptor, promoting an effect on cell proliferation as has been shown for PC12 cells. However, S-CS and S-DCS would sequester the protein, decreasing the GF signaling activity by depleting the protein or locally blocking its active site.

Efficient reduction of Toluidine Blue O dye using silver nanoparticles synthesized by low molecular weight chitosans.

The aim of this paper is to study the catalytic behaviour of silver nanoparticles (AgNps) produced using low molecular weight chitosan (LMWC) samples depolymerized by an enzymatic method, using either lysozyme or chitosanase. The ability of four sets of silver nanoparticles to reduce Toluidine Blue (TBO) was used as test reaction, and the effect of both catalyst concentration and reaction temperature on the effectiveness of the catalytic reduction was assessed. Generally speaking, AgNps produced through chitosan depolymerization with lysozyme showed better performance than those ones produced using chitosanase. On the other hand, colloidal silver nanoparticles stabilized with LMWC were mixed with medium molecular weight chitosan (MMWC) sample, in order to generate different scaffolds by using beta-glycerol phosphate. These scaffolds were analyzed by microscopy, XRD, ATR-FTIR, and their swelling capacity was evaluated. Their catalytic ability for reducing TBO, as well as their reusability, was assessed. Our results showed that the catalytic properties of the colloidal AgNps were remarkably affected by the properties of the LMWC used for their synthesis. Once again, AgNps-chitosan scaffolds produced with chitosan depolymerized with lysozyme were more effective.

Preparation of a crude chitosanase from blue crab viscera as well as its application in the production of biologically active chito-oligosaccharides from shrimp shells chitosan.

In this study, a digestive chitosanase from blue crab (Portunus segnis) viscera was extracted, characterized and applied. The crude chitosanase showed optimum activity at pH 4.0 and 60 °C and retained >80% of its activity over a pH range from 3.0 to 10.0. Subsequently, the crude chitosanase was applied to produce bioactive varying molecular weight (Mw) and acetylation degree chitosan-depolymerization products (CDP) with specially sequences composition determined by MALDI-TOF MS owing to an endo-cleavage mode. This hydrolysis process allowed to the preparation, after 24 h of incubation at 40 °C, of a low Mw water soluble CDP (H 24h, <4.4 kDa) with DP up to 6 and a high Mw CDP (C 24h, 142.19 kDa). Following their physicochemical characterization, the functional properties, antioxidant and antimicrobial activities of CDP were investigated. Interestingly, as compared to the native chitosan, CDP, especially low Mw derivatives (H 24h) exhibited potent antioxidant activities, while high Mw derivatives, especially C 24h, markedly inhibited the growth of all tested bacteria and fungi. These results may provide novel insights into the efficiency of chitosan depolymerisation using the Portunus segnis digestive crude chitosanase as a simple, inexpensive and easily method to produce bioactive chitosan-derivatives and that this bioactivity depends highly on their attractive characteristics.

New drug delivery systems based on chitosan.

The development of new delivery systems for the controlled release of drugs is one of the most interesting fields of research in pharmaceutical sciences. Microparticles can be used for the controlled release of drugs, vaccines, antibiotics, and hormones. To prevent the loss of encapsulated materials, the microcapsules should be coated with another polymer that forms a membrane on the surface. In addition there are several fundamental properties of polymers that are useful in solving drug delivery problems, as they can be combined with the drug covalently or ionically to overcome problems like solubility, stability or permeability. Chitosan is a copolymer of N-acetylglucosamine and glucosamine derived from chitin, which is extracted from crustaceans' shells. This polymer has become the focus of major interest in recent years because it has applications in several fields such as biomedicine, agriculture, the textile industry and the paper industry. There are many processes that can be used to encapsulate drugs within chitosan matrixes such as ionotropic gelation, spray drying, emulsification-solvent evaporation and coacervation. Combinations of these processes are also used in order to obtain microparticles with specific properties and performances. This review provides an overview of these four techniques applied directly to chitosan microparticulate systems.

On the Ability of Low Molecular Weight Chitosan Enzymatically Depolymerized to Produce and Stabilize Silver Nanoparticles.

Silver nanoparticles (AgNPs) are of great interest due to their antimicrobial, optical and catalytical properties. Green synthesis of AgNPs is fundamental for some applications such as biomedicine and catalysis. Natural polymers, such as chitosan, have been proposed as reducing and stabilizing agents in the green synthesis of AgNPs. Physico-chemical properties of chitosan have a great impact on its technological and biological properties. In this paper, we explore the effect of chitosan molecular weight (Mw) on the thermal AgNPs production using two sample sets of low Mw chitosans (F1 > 30 kDa, F2: 30⁻10 kDa and F3: 10⁻5 kDa) produced by enzymatic depolymerization of a parent chitosan with chitosanase and lysozyme. Both polymer sets were able to effectively reduce Ag+ to Ag0 as the presence of the silver surface plasmon resonance (SRP) demonstrated. However, the ability to stabilize the nanoparticles depended not only on the Mw of the polymer but particularly on the polymer pattern which was determined by the enzyme used to depolymerize the parent chitosan. Low Mw chitosan samples produced by lysozyme were more effective than those produced by chitosanase to stabilize the AgNPs and smaller and less polydisperse nanoparticles were visualized by transmission electron microscopy (TEM). With some polymer sets, more than 80% of the AgNPs produced were lower than 10 nm which correspond to quantum dots. The preparation method described in this paper is general and therefore, it may be extended to other noble metals, such as palladium, gold or platinum.

Synthesis, physicochemical characterization and biological evaluation of chitosan sulfate as heparan sulfate mimics.

Despite the relevant biological functions of heparan sulfate (HS) glycosaminoglycans, their limited availability and the chemical heterogeneity from natural sources hamper their use for biomedical applications. Chitosan sulfates (ChS) exhibit structural similarity to HSs and may mimic their biological functions. We prepared a variety of ChS with different degree of sulfation to evaluate their ability to mimic HS in protein binding and to promote neural cell division and differentiation. The structure of the products was characterized using various spectroscopic and analytical methods. The study of their interaction with different growth factors showed that ChS bound to the proteins similarly or even better than heparin. In cell cultures, a transition effect on cell number was observed as a function of ChS concentration. Differences in promoting the expression of the differentiation markers were also found depending on the degree of sulfation and modification in the chitosan.

Effect of chito-oligosaccharides over human faecal microbiota during fermentation in batch cultures.

Chitosan with high number of deacetylated units, its reacetylated derivative and COS obtained through an enzymatic treatment with chitosanase were tested in pH controlled batch cultures to investigate the ability of the human faecal microbiota to utilise them. Chitosan derivatives with high number of deacetylated units decreased the bacterial populations: Bifidobacterium spp., Eubacterium rectale/Clostridium coccoides, C. Histolyticum and Bacteroides/Prevotella. On the other hand, chitosan derivatives with high content of acetylated residues maintained the tested bacterial groups and could increase Lactobacillus/Enterococcus. Regarding short chain fatty acids (SCFA), only low Mw COS increased the production in similar levels than fructo-oligossacharides (FOS). The acetylated chitosans and their COS do not appear as potential prebiotics but did not affect negatively the faecal microbiota, while derivatives with high number of deacetylated units could induce a colonic microbiota imbalance.

Fluorescent imino and secondary amino chitosans as potential sensing biomaterials.

A variety of fluorescent imino and secondary amino chitosans were synthesized under very mild conditions by reaction of the biopolymer amino functions with aromatic aldehydes in an acidified methanolic suspension. Simultaneous reactions of several aldehydes with chitosan were successfully carried out, and kinetic studies showed that 1-pyrenecarboxaldehyde reacts the fastest among them. An unprecedented study on the evaluation of the degree of N-substitution (DS, ranging from 31.7% to 12.0%) for the chitosan Schiff bases by using solid state CPMAS (13)C NMR is performed. A linear correlation between the DS obtained for the secondary amino chitosans by (1)H NMR (55.3-10.2%) and those obtained by CPMAS (13)C NMR (34.4-13.8%) has allowed us to calculate an empirical correlation factor that could be applied on chitosan-based aromatic systems. The new chiral-labelled chitosan derivatives exhibit a stable fluorescent behaviour, which was used to explore solvent sensoring applications.

Influence of the physico-chemical characteristics of chito-oligosaccharides (COS) on antioxidant activity.

Chito-oligosaccharides (COS) are being used as important functional materials for many applications due to their bioactivities. The aim of this research has been to assess the relationship between the physico-chemical characteristics, average molecular weight (Mw), acetylation degree (DA), polymerization degree (DP) and specially sequence composition determined by MALDI-TOF MS and the antioxidant properties of COS. These oligosaccharides were obtained by enzymatic depolymerization with chitosanase and lysozyme using a specific chitosan and its reacetylated product. The COS fraction below 5 kDa obtained from chitosanase depolymerization showed the highest capacity to scavenge DPPH radicals and to reduce Fe(3+). A correlation was found between the relative amount of molecules with a given A/D (acetylated vs deacetylated units) ratio within the COS and their antioxidant activity, which could be used to predict the antioxidant behavior of a fraction of chito-oligosaccharides.

Tight junction modulation by chitosan nanoparticles: comparison with chitosan solution.

Present work investigates the potential of chitosan nanoparticles, formulated by the ionic gelation with tripolyphosphate (TPP), to open the cellular tight junctions and in doing so, improve the permeability of model macromolecules. A comparison is made with chitosan solution at equivalent concentrations. Initial work assessed cytotoxicity (through MTS and LDH assays) of chitosan nanoparticles and solutions on Calu-3 cells. Subsequently, a concentration of chitosan nanoparticles and solution exhibiting minimal toxicity was used to investigate the effect on TEER and macromolecular permeability across filter-cultured Calu-3 monolayer. Chitosan nanoparticles and solution were also tested for their effect on the distribution of the tight junction protein, zonnula occludens-1 (ZO-1). Chitosan nanoparticles produced a sharp and reversible decrease in TEER and increased the permeability of two FITC-dextrans (FDs), FD4 (MW 4 kDa) and FD10 (MW 10 kDa), with effects of a similar magnitude to chitosan solution. Chitosan nanoparticles produced changes in ZO-1 distribution similar to chitosan solution, indicating a tight junction effect. While there was no improvement in permeability with chitosan nanoparticles compared to solution, nanoparticles provide the potential for drug incorporation, and hence the possibility for providing controlled drug release and protection from enzymatic degradation.