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.

Exploring Saduria entomon (Crustacea Isopoda) as a New Source for Chitin and Chitosan Isolation.

Chitin and chitosan demand is growing very fast due to interest from industries such as pharmaceutical, cosmetic, agricultural and others. New sources for chitin and chitosan isolation are being extensively searched to fulfil this demand. In this paper, Saduria entomon a Baltic benthic crustacean, is evaluated as a source for chitin and chitosan isolation. Chitin and chitosan yield from S. entomon were 14.8 and 8.2%, respectively, in a similar range to other sources. Samples were characterized in terms of physicochemical properties (acetylation degree, molecular weight, thermal stability, and crystallinity) and two biological properties, antimicrobial activity and antioxidant activity were evaluated. Chitosan S. entomon exhibited antimicrobial activity against S. aureus but not against E. coli. An antioxidant activity of 20.98 TROLOX µmol equivalent/g polymer was detected for the chitosan sample. These properties are very promising for the use of this organism as a source for chitin and chitosan isolation in the biomedical field.

Development of Methotrexate Complexes Endowed with New Biological Properties Envisioned for Musculoskeletal Regeneration in Rheumatoid Arthritis Environments.

Methotrexate (MTX) administration is the gold standard treatment for rheumatoid arthritis (RA), but its effects are limited to preventing the progression of the disease. Therefore, effective regenerative therapies for damaged tissues are still to be developed. In this regard, MTX complexes of general molecular formula M(MTX)·xH2O, where M = Sr, Zn, or Mg, were synthesized and physicochemically characterized by TGA, XRD, NMR, ATR-FTIR, and EDAX spectroscopies. Characterization results demonstrated the coordination between the different cations and MTX via two monodentate bonds with the carboxylate groups of MTX. Cation complexation provided MTX with new bioactive properties such as increasing the deposition of glycosaminoglycans (GAGs) and alternative anti-inflammatory capacities, without compromising the immunosuppressant properties of MTX on macrophages. Lastly, these new complexes were loaded into spray-dried chitosan microparticles as a proof of concept that they can be encapsulated and further delivered in situ in RA-affected joints, envisioning them as a suitable alternative to oral MTX therapy.

Short-Chain Chitin Oligomers: Promoters of Plant Growth.

Chitin is the second most abundant biopolymer in nature after cellulose, and it forms an integral part of insect exoskeletons, crustacean shells, krill and the cell walls of fungal spores, where it is present as a high-molecular-weight molecule. In this study, we showed that a chitin oligosaccharide of lower molecular weight (tetramer) induced genes in Arabidopsis that are principally related to vegetative growth, development and carbon and nitrogen metabolism. Based on plant responses to this chitin tetramer, a low-molecular-weight chitin mix (CHL) enriched to 92% with dimers (2mer), trimers (3mer) and tetramers (4mer) was produced for potential use in biotechnological processes. Compared with untreated plants, CHL-treated plants had increased in vitro fresh weight (10%), radicle length (25%) and total carbon and nitrogen content (6% and 8%, respectively). Our data show that low-molecular-weight forms of chitin might play a role in nature as bio-stimulators of plant growth, and they are also a known direct source of carbon and nitrogen for soil biomass. The biochemical properties of the CHL mix might make it useful as a non-contaminating bio-stimulant of plant growth and a soil restorer for greenhouses and fields.

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.

Cell Adhesion and Proliferation on Sulfonated and Non-Modified Chitosan Films.

Three types of chitosan-based films have been prepared and evaluated: a non-modified chitosan film bearing cationizable aliphatic amines and two films made of N-sulfopropyl chitosan derivatives bearing both aliphatic amines and negative sulfonate groups at different ratios. Cell adhesion and proliferation on chitosan films of C2C12 pre-myoblastic cells and B16 cells as tumoral model have been tested. A differential cell behavior has been observed on chitosan films due to their different surface modification. B16 cells have shown lower vinculin expression when cultured on sulfonated chitosan films. This study shows how the interaction among cells and material surface can be modulated by physicochemical characteristics of the biomaterial surface, altering tumoral cell adhesion and proliferation processes.

Preparation of chitosan nanocomposites with a macroporous structure by unidirectional freezing and subsequent freeze-drying.

Chitosan is the N-deacetylated derivative of chitin, a naturally abundant mucopolysaccharide that consists of 2-acetamido-2-deoxy-ß-d-glucose through a ß (1→4) linkage and is found in nature as the supporting material of crustaceans, insects, etc. Chitosan has been strongly recommended as a suitable functional material because of its excellent biocompatibility, biodegradability, non-toxicity, and adsorption properties. Boosting all these excellent properties to obtain unprecedented performances requires the core competences of materials chemists to design and develop novel processing strategies that ultimately allow tailoring the structure and/or the composition of the resulting chitosan-based materials. For instance, the preparation of macroporous materials is challenging in catalysis, biocatalysis and biomedicine, because the resulting materials will offer a desirable combination of high internal reactive surface area and straightforward molecular transport through broad "highways" leading to such a surface. Moreover, chitosan-based composites made of two or more distinct components will produce structural or functional properties not present in materials composed of one single component. Our group has been working lately on cryogenic processes based on the unidirectional freezing of water slurries and/or hydrogels, the subsequent freeze-drying of which produce macroporous materials with a well-patterned structure. We have applied this process to different gels and colloidal suspensions of inorganic, organic, and hybrid materials. In this review, we will describe the application of the process to chitosan solutions and gels typically containing a second component (e.g., metal and ceramic nanoparticles, or carbon nanotubes) for the formation of chitosan nanocomposites with a macroporous structure. We will also discuss the role played by this tailored composition and structure in the ultimate performance of these materials.

Chemical guiding of magnetic nanoparticles in dispersed media containing poly-(methylmethacrylate-co-vinylpyrrolidone).

The differential reactivity of methylmethacrylate (MMA) and vinylpyrrolidone (VP) in free radical copolymerization, with stirring in methanol, renders an emulsified two phase system. The dispersed and continuous liquid phases contain copolymers rich in MMA and VP, respectively. When Fe(3)O(4) magnetic nanoparticles (mNPs) stabilized with tetramethylammonium hydroxide are added to this emulsion, the mNPs are located in the continuous phase. Very small chemical changes in the methacrylic or vinylic chains are able to guide the mNP toward the interface or to the inside of the dispersed phase since quite a selective functionalization of each phase may be achieved separately. Thus, a small addition of methacrylic acid as comonomer (0.5% molar) guides all of the mNPs to the interface while a 0.5% molar of sulfopropyl methacrylate induces the migration of all mNPs to the dispersed phase. When 0.5% molar of a VP derivative bearing sulfonate functionality is added, the mNPs are found both in the interface and in the continuous phase. The addition of water allows solid MMA-based microspheres to be obtained incorporating the mNPs selectively either at the surface or in the core.

Evaluating Non-Conventional Chitosan Sources for Controlled Release of Risperidone.

In this work, two chitosan samples from cuttlebone and squid pen are produced and characterized. We studied the formation of thermoresponsive hydrogels with ß-glycerol phosphate and found proper formulations that form the hydrogels at 37 °C. Gel formation depended on the chitosan source being possible to produce the thermoresponsive hydrogels at chitosan concentration of 1% with cuttlebone chitosan but 1.5% was needed for squid pen. For the first time, these non-commercial chitosan sources have been used in combination with ß-glycerol phosphate to prepare risperidone formulations for controlled drug delivery. Three types of formulations for risperidone-controlled release have been developed, in-situ gelling formulations, hydrogels and xerogels. The release profiles show that in-situ gelling formulations and particularly hydrogels allow an extended control release of risperidone while xerogels are not appropriate formulations for this end since risperidone was completely released in 48 h.

Chitosan gelation induced by the in situ formation of gold nanoparticles and its processing into macroporous scaffolds.

This work describes a simple synthetic route to induce chitosan (CHI) gelation by the in situ formation of gold nanoparticles (AuNPs). AuNPs were obtained by thermal treatment (e.g., 40 and 80 °C) of CHI aqueous solutions containing HAuCl(4) and in the absence of further reducing agents. The CHI hydrogels resulting after AuNP formation were submitted to unidirectional freezing and subsequent freeze-drying via ISISA (ice-segregation-induced self-assembly) process for the preparation of CHI scaffolds. The study of AuNP-CHI scaffolds by SEM and confocal fluorescence microscopy revealed a morphological structure characteristic of the hydrogel nature of the samples subjected to the ISISA process. Interestingly, not only the morphology but also the dissolution and swelling degree of the resulting CHI scaffolds were strongly influenced by the strength of the hydrogels obtained by the in situ formation of AuNP. We have also studied the catalytic activity AuNP-CHI scaffolds in the reduction of p-nitrophenol. The negligible dissolution and low swelling degree obtained in certain AuNP-CHI scaffolds allowed them to be used for more than four cycles with full preservation of the reaction kinetics.

Chitin- and Chitosan-Based Composite Materials.

Chitin and its deacetylated derivative chitosan are amino polysaccharides of great interest due to their biological and technological properties [...].

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: An Overview of Its Properties and Applications.

Chitosan has garnered much interest due to its properties and possible applications. Every year the number of publications and patents based on this polymer increase. Chitosan exhibits poor solubility in neutral and basic media, limiting its use in such conditions. Another serious obstacle is directly related to its natural origin. Chitosan is not a single polymer with a defined structure but a family of molecules with differences in their composition, size, and monomer distribution. These properties have a fundamental effect on the biological and technological performance of the polymer. Moreover, some of the biological properties claimed are discrete. In this review, we discuss how chitosan chemistry can solve the problems related to its poor solubility and can boost the polymer properties. We focus on some of the main biological properties of chitosan and the relationship with the physicochemical properties of the polymer. Then, we review two polymer applications related to green processes: the use of chitosan in the green synthesis of metallic nanoparticles and its use as support for biocatalysts. Finally, we briefly describe how making use of the technological properties of chitosan makes it possible to develop a variety of systems for drug delivery.

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.

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.

Ionic Conductivity, Diffusion Coefficients, and Degree of Dissociation in Lithium Electrolytes, Ionic Liquids, and Hydrogel Polyelectrolytes.

The conductive and diffusional behavior of electrolytes in media with different dielectric and viscoelastic properties is investigated. A revised model to separate the contribution of dissociated and nondissociated species to the diffusion coefficients determined with NMR is proposed. Impedance spectroscopy is used to measure the ionic conductivity of lithium salts in aqueous medium, ionic liquids in aprotic solvents, and hydrogel polyelectrolytes. The diffusion coefficients of the species of interest in those systems are determined with multinuclear pulsed-gradient spin-echo (PGSE) NMR. The results are analyzed using the revised model. It is shown that the degree of ionization could be determined directly from measurements of ionic conductivity and diffusion coefficients in very different types of electrolytes and in a wide range of concentrations. Furthermore, these findings support the original Arrhenius hypothesis about electrolytes and show that the assumption of a complete dissociation is not required to describe their conductive behavior. The reduced conductivity observed in hydrogels, at or near swelling equilibrium, compared to that in solutions could be attributed mainly to the hindered ionic mobility caused by the network structure.