Systematic Structural Characterization of Chitooligosaccharides Enabled by Automated Glycan Assembly.

Chitin, a polymer composed of ß(1-4)-linked N-acetyl-glucosamine monomers, and its partially deacetylated analogue chitosan, are abundant biopolymers with outstanding mechanical as well as elastic properties. Their degradation products, chitooligosaccharides (COS), can trigger the innate immune response in humans and plants. Both material and biological properties are dependent on polymer length, acetylation, as well as the pH. Without well-defined samples, a complete molecular description of these factors is still missing. Automated glycan assembly (AGA) enabled rapid access to synthetic well-defined COS. Chitin-cellulose hybrid oligomers were prepared as important tools for a systematic structural analysis. Intramolecular interactions, identified by molecular dynamics simulations and NMR analysis, underscore the importance of the chitosan amino group for the stabilization of specific geometries.

Chitosan chemically modified to deliver nitric oxide with high antibacterial activity.

The aim of the current study is to report a simple and efficient method to chemically modify chitosan in order to form S-nitroso-chitosan for antibacterial applications. Firstly, commercial chitosan (CS) was modified to form thiolated chitosan (TCS) based on an easy and environmental-friendly method. TCS was featured based on physicochemical and morphological techniques. Results have confirmed that thiol groups in TCS formed after CS's primary amino groups were replaced with secondary amino groups. Free thiol groups in TCS were nitrosated to form S-nitrosothiol moieties covalently bond to the polymer backbone (S-nitroso-CS). Kinetic measurements have shown that S-nitroso-CS was capable of generating NO in a sustained manner at levels suitable for biomedical applications. The antibacterial activities of CS, TCS and S-nitroso-CS were evaluated based on the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill curves determined for Escherichia coli, Staphylococcus aureus and Streptococcus mutans. MIC/MBC values reached 25/25, 0.7/0.7 and 3.1/3.1 µg mL-1 for CS/TCS and 3.1/3.1, 0.1/0.2, 0.1/0.2 µg mL-1 for S-nitroso-CS, respectively. Decreased MIC and MBC values have indicated that S-nitroso-CS has higher antibacterial activity than CS and TCS. Time-kill curves have shown that the bacterial cell viability decreased 5-fold for E. coli and 2-fold for S. mutans in comparison to their respective controls, after 0.5 h of incubation with S-nitroso-CS. Together, CS backbone chemically modified with S-nitroso moieties have yielded a polymer capable of generating therapeutic NO concentrations with strong antibacterial effect.

Thiomers of Chitosan and Cellulose: Effective Biosorbents for Detection, Removal and Recovery of Metal Ions from Aqueous Medium.

Removal of toxic metal ions using adsorbents is a well-known strategy for water treatment. While chitosan and cellulose can adsorb weakly some types of metals, incorporating thiols as metal chelating agents can improve their sorption behaviors significantly. Presented in this review are the various chemical modification strategies applicable for thiolation of chitosan and cellulose in the forms of mercaptans, xanthates and dithiocarbamates. Moreover, much attention has been paid to the specific strategies for controlling the thiolation degree and characterization approaches for establishing the structure-property relationship. Also, the kinetics and isotherm models that elucidate the adsorption processes and mechanisms induced by the thiomers have been explained. These thiomers have found great potentials in the applications associated with metal removal, metal recovery and metal detection.

Polymorphism of chitosan-based networks stabilized by phytate investigated by molecular dynamics simulations.

Chitosan can associate in the presence of polyphosphates into insoluble hydrogels capable of drug encapsulation and safe and efficient release. On the one hand, chitosan hydrogels were synthesized using the phytate anion as a crosslinking agent and were characterized by employing dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR). On the other hand, an effective chitosan-phytate model with atomistic details was created to examine the underlying physical crosslinking pattern, and the structure and dynamics of the chitosan-phytate complex were systematically investigated by using molecular dynamics (MD) simulations. To harbor the crosslinker potential for obtaining chitosan-based hydrogels, the impact of the phytate concentration and the functional groups of the chitosan on the reticulation process was addressed. The phytate association was determined by the phosphates' capacity for H-bonding to the amine and hydroxyl groups belonging to two consecutive glucosidic units. The physical crosslinking pattern was determined by the number of chitosan chains bound by one phytate anion and the phytate orientation relative to the glucopyranose neighbors. Cross-linking of two up to six chitosan chains mediated by a phytate anion represented favorable states, and the number distribution of cross-linked chains depended on the phytate concentration. The circular distribution of the cross-linkable phosphates regulated the nearly isotropic orientation of the chitosan chains and phytate at the junction, and the variety of topological crosslinking demonstrated the phytate ion's potential for developing chitosan-based hydrogels with improved structural attributes.

Spray Drying of Chitosan Acid Salts: Process Development, Scaling Up and Physicochemical Material Characterization.

We investigated a spray drying process for preparing water-soluble salts of high molecular weight chitosan (CH) intended for pharmaceutical excipient applications. CH was derived from chitin of marine lobster origin (Panulirus argus). The effects of organic acid (acetic or lactic acid) and the ratio (difference) of inlet/outlet air temperature (140/90 °C or 160/100 °C) on spray drying were studied. The yield of spray-dried CH salt powders ranged from 50% to 99% in laboratory and industrial-scale processes. The spray-dried dry powder of CH salts consisted of spherical agglomerated particles with an average diameter of 36.2 ± 7.0 µm (CH acetate) and 108.6 ± 11.5 µm (CH lactate). After dispersing the spray-dried CH salt powder samples in purified water, the mean particle sizes obtained for the CH acetate salts were 31.4 nm (batch A001), 33.0 nm (A002) and 44.2 nm (A003), and for the CH lactate salts 100.8 nm (batch L001), 103.2 nm (L002) and 121.8 nm (L003). The optimum process conditions for spray drying were found: an inlet air temperature of 160 ± 5 °C, an outlet temperature of 100 ± 5 °C and an atomizer disk rotational speed of 18,200 min-1. The X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) results confirmed the amorphous state of the CH salts. The 1H nuclear magnetic resonance (NMR) and Fourier transform infrared (FT-IR) spectra of CH acetate and lactate salts verified that the spray drying process does not affect the polymer backbone. In conclusion, both laboratory and industrial-scale spray drying methods for preparing water-soluble acid salts of CH are reproducible, and the physicochemical properties of the corresponding CH acid salts are uniform.

Design and Preclinical Evaluation of Chitosan/Kaolin Nanocomposites with Enhanced Hemostatic Efficiency.

In the current study, hemostatic compositions including a combination of chitosan and kaolin have been developed. Chitosan is a marine polysaccharide derived from chitins, a structural component in the shells of crustaceans. Both chitosan and kaolin have the ability to mediate a quick and efficient hemostatic effect following immediate application to injury sites, and thus they have been widely exploited in manufacturing of hemostatic composites. By combining more than one hemostatic agent (i.e., chitosan and kaolin) that act via more than one mechanism, and by utilizing different nanotechnology-based approaches to enhance the surface areas, the capability of the dressing to control bleeding was improved, in terms of amount of blood loss and time to hemostasis. The nanotechnology-based approaches utilized to enhance the effective surface area of the hemostatic agents included the use of Pluronic nanoparticles, and deposition of chitosan micro- and nano-fibers onto the carrier. The developed composites effectively controlled bleeding and significantly improved hemostasis and survival rates in two animal models, rats and rabbits, compared to conventional dressings and QuikClot® Combat Gauze. The composites were well-tolerated as demonstrated by their in vivo biocompatibility and absence of clinical and biochemical changes in the laboratory animals after application of the dressings.

The improved antiviral activities of amino-modified chitosan derivatives on Newcastle virus.

Chitosan is widely used as a medical material because of its excellent biological activities. However, the low solubility of natural chitosan limited its medicinal activity to some extent. The solubility can be improved by introducing more active groups and lowering molecular weight. Therefore, 6-amine chitosan derivatives were synthesized in this paper since more active groups were introduced to increase the medicinal activity. Those derivatives were characterized by elemental analysis, HPLC, and FT-IR and the antiviral activity was tested by hemagglutination tests. Finally, 6-amine chitosan derivatives improved the antiviral activity, especially after the introduction of bromine ion. When 6-deoxy-6-bromo-N-phthaloyl chitosan was 1 g/L, they reduced the hemagglutination titer of virus to zero. The RT-PCR result showed that the expression level of TNF-α and IFN-ß increased significantly, which indicated that the antiviral activity of amino-modified chitosan worked through the stimulation of immune response.

Evaluation of hyaluronic acid nanoparticle embedded chitosan-gelatin hydrogels for antibiotic release.

The development of chitosan-gelatin (CS-G) hydrogels embedded with ampicillin-loaded hyaluronic acid nanoparticles (HA-NPs) for wound dressing is proposed. It was aimed to provide controlled ampicillin delivery by incorporation of HA-NPs into biocompatible CS-G hydrogel structure. According to in vitro ampicillin release studies, 55% of ampicillin was released from CS-G/HA-NPs hydrogels after 5 days. Antibacterial performance of CS-G/HA-NPs hydrogels was proven with agar disc diffusion test. For cytotoxicity assay, fibroblast cell viability increased in CS-G/HA-NPs hydrogels compared with CS-G group after 24 hr incubation. Consequently, the potential ability of CS-G/HA-NPs hydrogels as a controlled drug delivery system has been verified.

A Novel Carbon Dots/Thermo-Sensitive In Situ Gel for a Composite Ocular Drug Delivery System: Characterization, Ex-Vivo Imaging, and In Vivo Evaluation.

We developed a potential composite ocular drug delivery system for the topical administration of diclofenac sodium (DS). The novel carbon dot CDC-HP was synthesized by the pyrolysis of hyaluronic acid and carboxymethyl chitosan through a one-step hydrothermal method and then embedded in a thermosensitive in situ gel of poloxamer 407 and poloxamer 188 through swelling loading. The physicochemical characteristics of these carbon dots were investigated. The results of the in vitro release test showed that this composite ocular drug delivery system (DS-CDC-HP-Gel) exhibited sustained release for 12 h. The study of the ex vivo fluorescence distribution in ocular tissues showed that it could be used for bioimaging and tracing in ocular tissues and prolong precorneal retention. Elimination profiles in tears corresponded to the study of ex vivo fluorescence imaging. The area under the curve of DS in the aqueous humor in the DS-CDC-HP-Gel group was 3.45-fold that in the DS eye drops group, indicating a longer precorneal retention time. DS-CDC-HP with a positive charge and combined with a thermosensitive in situ gel might strengthen adherence to the corneal surface and prolong the ocular surface retention time to improve the bioavailability. This composite ocular delivery system possesses potential applications in ocular imaging and drug delivery.

Bio-Functionalized Chitosan for Bone Tissue Engineering.

Hybrid biomaterials allow for the improvement of the biological properties of materials and have been successfully used for implantology in medical applications. The covalent and selective functionalization of materials with bioactive peptides provides favorable results in tissue engineering by supporting cell attachment to the biomaterial through biochemical cues and interaction with membrane receptors. Since the functionalization with bioactive peptides may alter the chemical and physical properties of the biomaterials, in this study we characterized the biological responses of differently functionalized chitosan analogs. Chitosan analogs were produced through the reaction of GRGDSPK (RGD) or FRHRNRKGY (HVP) sequences, both carrying an aldehyde-terminal group, to chitosan. The bio-functionalized polysaccharides, pure or "diluted" with chitosan, were chemically characterized in depth and evaluated for their antimicrobial activities and biocompatibility toward human primary osteoblast cells. The results obtained indicate that the bio-functionalization of chitosan increases human-osteoblast adhesion (p < 0.005) and proliferation (p < 0.005) as compared with chitosan. Overall, the 1:1 mixture of HVP functionalized-chitosan:chitosan is the best compromise between preserving the antibacterial properties of the material and supporting osteoblast differentiation and calcium deposition (p < 0.005 vs. RGD). In conclusion, our results reported that a selected concentration of HVP supported the biomimetic potential of functionalized chitosan better than RGD and preserved the antibacterial properties of chitosan.

Grafting of 18ß-Glycyrrhetinic Acid and Sialic Acid onto Chitosan to Produce a New Amphipathic Chitosan Derivative: Synthesis, Characterization, and Cytotoxicity.

Chitosan is the only cationic polysaccharide found in nature. It has broad application prospects in biomaterials, but its application is limited due to its poor solubility in water. A novel chitosan derivative was synthesized by amidation of chitosan with 18ß-glycyrrhetinic acid and sialic acid. The chitosan derivatives were characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, and measurement of the zeta potential. We also investigated the solubility, cytotoxicity, and blood compatibility of chitosan derivatives. 18ß-glycyrrhetinic acid and sialic acid could be grafted onto chitosan molecular chains. The thermal stability of the synthesized chitosan derivatives was decreased and the surface was positively charged in water and phosphate-buffered saline. After chitosan had been modified by 18 ß-glycyrrhetinic acid and sialic acid, the solubility of chitosan was improved greatly in water and phosphate-buffered saline, and percent hemolysis was <5%. Novel amphiphilic chitosan derivatives could be suitable polymers for biomedical purposes.

Nanoparticles integrating natural and synthetic polymers for in vivo insulin delivery.

The aims of the current study were to develop insulin-loaded nanoparticles comprised of various polymers at different compositions, and to evaluate their ability to lower blood glucose levels in diabetic rats following subcutaneous and oral administrations. Several combinations of natural and synthetic polymers have been utilized for preparation of nanoparticles including, chitosan, alginate, albumin and Pluronic. Nanosized (170 nm-800 nm) spherical particles of high encapsulation efficiency (15-52%) have been prepared. Composition and ratios between the integrated polymers played a pivotal role in determining size, zeta potential, and in vivo hypoglycemic activity of particles. After subcutaneous and oral administration in diabetic rats, some of the insulin-loaded nanoparticles were able to induce much higher hypoglycemic effect as compared to the unloaded free insulin. For instance, subcutaneous injection of nanoparticles comprised of chitosan combined with sodium tripolyphosphate, Pluronic or alginate/calcium chloride, resulted in comparable hypoglycemic effects to free insulin, at two-fold lower dose. Nanoparticles were well-tolerated after oral administration in rats, as evidenced by by measuring levels of alanine aminotransferase, aspartate aminotransferases, albumin, creatinine and urea. This study indicates that characteristics and delivery efficiency of nanomaterials can be controlled via utilizing several natural/synthetic polymers and by fine-tuning of combination ratio between polymers.

Study of the Acetylation Pattern of Chitosan by Pure Shift NMR.

Chitosan is a biodegradable, antibacterial, and nontoxic biopolymer used in a wide range of applications including biotechnology, pharmacy, and medicine. The physicochemical and biological properties of chitosan have been associated with parameters such as the degree of polymerization (DP) and the fraction of acetylation (FA). New methods are being developed to yield chitosans of specific acetylation patterns, and, recently, a correlation between biological activity and the distribution of the acetylated units (PA: pattern of acetylation) has been demonstrated. Although there are numerous well-established methods for the determination of DP and FA values, this is not the case for PA. The methods available are either not straightforward or not sensitive enough, limiting their use for routine analysis. In this study, we demonstrate that by applying HOmodecoupled Band-Selective (HOBS) decoupling NMR on signals assigned by multidimensional Pure Shift NMR methods, PA can be easily and accurately determined on various chitosan samples. This novel methodology-easily implemented for routine analysis-could become a standard for chitosan PA assessment. In addition, by applying Spectral Aliased Pure Shift HSQC, the analysis was enhanced with the determination of triads.

Green and Functional Aerogels by Macromolecular and Textural Engineering of Chitosan Microspheres.

Tremendous interest was recently devoted to the preparation of porous and functional materials through sustainable route, including primarily the use of renewable biopolymers instead of petroleum-sourced synthetic chemicals. Among the biopolymers available in enormous quantity, chitosan - obtained by deacetylation of chitin - stands as the sole nitrogen-containing cationic amino-sugar carbohydrate. This distinctively provides chitosan derivatives with plenty of opportunities in materials science. Particularly, its pH switchable solubility allowed the preparation of three-dimensional entangled nanofibrillated self-standing microspheres. These porous hydrogels behave as nano-reactors to confine exogenous nanoobjects within the polysaccharide network, including sol-gel metal alkoxide species, organometallic derivatives and isotropic and oriented nanoparticles. Besides, the interfacial interplay of chitosan with lamellar clay and graphene oxide allowed the penetration of the biopolymer inside of the galleries, which result in a complete delamination of the layered nanomaterials. The preserved gelation memory of chitosan in these formulations provides a way to access porous microspheres entangling exfoliated nanometric sheets. CO2 supercritical drying of functional hydrogel beads enabled efficient removal of water and other liquid solvents without wall collapsing, allowing large-scale preparation of millimetric hydrocolloidal microspheres with an open macroporous network. These functionalized lightweight biopolymer aerogels find applications in heterogeneous catalysis, sensing, adsorption, insulation and for the design of other sophisticated porous nanostructures. Beyond their tailorable molecular and textural-engineering, the possibility for macroscopic shaping of these intriguing nanostructures opens many new opportunities, especially in additive-manufacturing for soft and hybrid robotics.

Development of Plasmonic Chitosan-Squarate Hydrogels via Bioinspired Nanoparticle Growth.

We report on the bioinspired growth of gold nanoparticles (GNPs) in biocompatible hydrogels to develop plasmonic hybrid materials. The new hydrogel (CS-Sq) is prepared from chitosan and diethylsquarate and is formed via noncovalent interactions rising between the in situ formed ionic squaric acid derivatives and chitosan. Interestingly, when the hydrogel is prepared in the presence of HAuCl4, GNPs with controlled sizes between 15 and 50 nm are obtained, which are homogeneously distributed within the plasmonic hydrogels (GNPs-CS-Sq). We found that the supramolecular nature and the composition of the CS-Sq hydrogels are key for the growth process of GNPs where the squaric derivatives act as reducing agents and the chitosan hydrogel network provides nucleation points and supports the GNPs. Accordingly, the hydrogel acts as a bioinspired reactor and permits to gain certain control on the size of GNPs by adjusting the concentration of chitosan and HAuCl4. Besides the intrinsic and tunable plasmonic properties of the GNPs-CS-Sq hydrogels, it was found that the gels could be useful as heterogeneous catalysts for organic reactions. Furthermore, cell viability studies indicate that the new hydrogels exhibit suitable biocompatibility. Thus, the proposed method for obtaining GNPs-CS-Sq hydrogels has the potential for the development of a wide variety of other hybrid chitosan materials useful for catalysis, biosensing, cell culture, tissue engineering, and drug delivery applications.

Tert-butyldimethylsilyl chitosan synthesis and characterization by analytical ultracentrifugation, for archaeological wood conservation.

The Oseberg ship is one of the most important archaeological testimonies of the Vikings. After excavation in 1904, the wooden gravegoods were conserved using alum salts. This resulted in extreme degradation of a number of the objects a hundred years later through acid depolymerisation of cellulose and lignin. The fragile condition of the artefacts requires a reconsolidation which has to be done avoiding water as solvent. We synthesized tert-butyldimethylsilyl (TBDMS) chitosan which is soluble in a 50:50 solution of ethyl acetate and toluene. Measurement of its molecular weight, to anticipate its penetration, provided a challenge as the density difference of the polymer and solvent was too small to provide adequate solute redistribution under a centrifugal field, so a two-stage process was implemented (i) determination of the weight-average molar mass of the aqueous soluble activated precursor, chitosan mesylate, Mw,mc using sedimentation equilibrium with the SEDFIT-MSTAR algorithm, and determination of the degree of polymerisation DP; (ii) measurement of the average degree of substitution DSTBDMS of the TBDMS group on each chitosan monosaccharide monomer unit using NMR, to augment the Mw,mc value to give the molar mass of the TBDMS-chitosan. For the preparation, we find Mw = 9.8 kg·mol-1, which is within the acceptable limit for penetration and consolidation of degraded wood. Future work will test this on archaeological wood from different sources.

On the Mechanism of Genipin Binding to Primary Amines in Lactose-Modified Chitosan at Neutral pH.

The present manuscript deals with the elucidation of the mechanism of genipin binding by primary amines at neutral pH. UV-VIS and CD measurements both in the presence of oxygen and in oxygen-depleted conditions, combined with computational analyses, led to propose a novel mechanism for the formation of genipin derivatives. The indications collected with chiral and achiral primary amines allowed interpreting the genipin binding to a lactose-modified chitosan (CTL or Chitlac), which is soluble at all pH values. Two types of reaction and their kinetics were found in the presence of oxygen: (i) an interchain reticulation, which involves two genipin molecules and two polysaccharide chains, and (ii) a binding of one genipin molecule to the polymer chain without chain-chain reticulation. The latter evolves in additional interchain cross-links, leading to the formation of the well-known blue iridoid-derivatives.

Ionotropic Gelation of Chitosan for Next-Generation Composite Proton Conducting Flat Structures.

(1) Background: Ionotropic gelation of cost-effective and eco-friendly biopolymer chitosan (Chit) is a novel and promising approach to the one-step synthesis of proton-conducting fuel cell bio-membranes.The method discovered by the author in 2011 and subsequently drowned among very few papers. This work aimed to relaunch this method through clear and effective communication of new unpublished results emphasizing the key aspects of this topic for successful dissemination of the results and significant future developments. (2) Methods and results: The mechanism of in-situ ionotropic gelation of Chit on an alumina substrate by phosphotungtate anions (PWA3-) was discussed and analyzed. The study sheds light on the effect of prolonged post-treatment in phosphotungstic acid (PWA) solution on the obtained chitosan/phosphotungstate (Chit-PWA) flat structures. Methods used included combined structural (XRD), thermal-gravimetric (DTG), electrochemical (in-situ EIS), compositional (EDX),morphological analysis (SEM), as well as the performances in a low temperature H2/O2 fuel cell(4) Conclusions: This contribution discloses novel possibilities aimed at increasing the impact of ionotropic gelation of chitosan on the scientific community working on the synthesis of novel proton conductive bio-composite membranes and structures.

Glycosylated-Chitosan Derivatives: A Systematic Review.

Chitosan derivatives, and more specifically, glycosylated derivatives, are nowadays attracting much attention within the scientific community due to the fact that this set of engineered polysaccharides finds application in different sectors, spanning from food to the biomedical field. Overcoming chitosan (physical) limitations or grafting biological relevant molecules, to mention a few, represent two cardinal strategies to modify parent biopolymer; thereby, synthetizing high added value polysaccharides. The present review is focused on the introduction of oligosaccharide side chains on the backbone of chitosan. The synthetic aspects and the effect on physical-chemical properties of such modifications are discussed. Finally, examples of potential applications in biomaterials design and drug delivery of these novel modified chitosans are disclosed.

Bioinspired Green Synthesis of Chitosan and Zinc Oxide Nanoparticles with Strong Antibacterial Activity against Rice Pathogen Xanthomonas oryzae pv. oryzae.

Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most devastating diseases, resulting in significant yield losses in rice. The extensive use of chemical antibacterial agents has led to an increase the environmental toxicity. Nanotechnology products are being developed as a promising alternative to control plant disease with low environmental impact. In the present study, we investigated the antibacterial activity of biosynthesized chitosan nanoparticles (CSNPs) and zinc oxide nanoparticles (ZnONPs) against rice pathogen Xoo. The formation of CSNPs and ZnONPs in the reaction mixture was confirmed by using UV-vis spectroscopy at 300-550 nm. Moreover, CSNPs and ZnONPs with strong antibacterial activity against Xoo were further characterized by scanning and transmission electron microscopy, Fourier-transform infrared spectroscopy, and X-ray diffraction. Compared with the corresponding chitosan and ZnO alone, CSNPs and ZnONPs showed greater inhibition in the growth of Xoo, which may be mainly attributed to the reduction in biofilm formation and swimming, cell membrane damage, reactive oxygen species production, and apoptosis of bacterial cells. Overall, this study revealed that the two biosynthesized nanoparticles, particularly CSNPs, are a promising alternative to control rice bacterial disease.