Applications of Chitosan in Surgical and Post-Surgical Materials

The continuous advances in surgical procedures require continuous research regarding materials with surgical applications. Biopolymers are widely studied since they usually provide a biocompatible, biodegradable, and non-toxic material. Among them, chitosan is a promising material for the development of formulations and devices with surgical applications due to its intrinsic bacteriostatic, fungistatic, hemostatic, and analgesic properties. A wide range of products has been manufactured with this polymer, including scaffolds, sponges, hydrogels, meshes, membranes, sutures, fibers, and nanoparticles. The growing interest of researchers in the use of chitosan-based materials for tissue regeneration is obvious due to extensive research in the application of chitosan for the regeneration of bone, nervous tissue, cartilage, and soft tissues. Chitosan can serve as a substance for the administration of cell-growth promoters, as well as a support for cellular growth. Another interesting application of chitosan is hemostasis control, with remarkable results in studies comparing the use of chitosan-based dressings with traditional cotton gauzes. In addition, chitosan-based or chitosan-coated surgical materials provide the formulation with antimicrobial activity that has been highly appreciated not only in dressings but also for surgical sutures or meshes.     

Stability of Chitosan—A Challenge for Pharmaceutical and Biomedical Applications

Chitosan—one of the natural multifunctional polymers—due to its unique and versatile biological properties is regarded as a useful compound in medical and pharmaceutical technology. Recently, considerable research effort has been made in order to develop safe and efficient chitosan products. However, the problem of poor stability of chitosan-based systems restricts its practical applicability; thus, it has become a great challenge to establish sufficient shelf-life for chitosan formulations. Improved stability can be assessed by controlling the environmental factors, manipulating processing conditions (e.g., temperature), introducing a proper stabilizing compound, developing chitosan blends with another polymer, or modifying the chitosan structure using chemical or ionic agents. This review covers the influence of internal, environmental, and processing factors on the long-term stability of chitosan products. The aim of this paper is also to highlight the latest developments which enable the physicochemical properties of chitosan-based applications to be preserved upon storage.     

Physicochemical and Antimicrobial Properties of Thermosensitive Chitosan Hydrogel Loaded with Fosfomycin

Thermosensitive chitosan hydrogels—renewable, biocompatible materials—have many applications as injectable biomaterials for localized drug delivery in the treatment of a variety of diseases. To combat infections such as Staphylococcus aureus osteomyelitis, localized antibiotic delivery would allow for higher doses at the site of infection without the risks associated with traditional antibiotic regimens. Fosfomycin, a small antibiotic in its own class, was loaded into a chitosan hydrogel system with varied beta-glycerol phosphate (β-GP) and fosfomycin (FOS) concentrations. The purpose of this study was to elucidate the interactions between FOS and chitosan hydrogel. The Kirby Bauer assay revealed an unexpected concentration-dependent inhibition of S. aureus, with reduced efficacy at the high FOS concentration but only at the low β-GP concentration. No effect of FOS concentration was observed for the planktonic assay. Rheological testing revealed that increasing β-GP concentration increased the storage modulus while decreasing gelation temperature. NMR showed that FOS was removed from the liquid portion of the hydrogel by reaction over 12 h. SEM and FTIR confirmed gels degraded and released organophosphates over 5 days. This work provides insight into the physicochemical interactions between fosfomycin and chitosan hydrogel systems and informs selection of biomaterial components for improving infection treatment.     

The Effect of Molecular Weight on the Antimicrobial Activity of Chitosan from Loligo opalescens for Food Packaging Applications

The growing requirement for sustainable processes has boosted the development of biodegradable plastic-based materials incorporating bioactive compounds obtained from waste, adding value to these products. Chitosan (Ch) is a biopolymer that can be obtained by deacetylation of chitin (found abundantly in waste from the fishery industry) and has valuable properties such as biocompatibility, biodegradability, antimicrobial activity, and easy film-forming ability. This study aimed to produce and characterize poly(lactic acid) (PLA) surfaces coated with β-chitosan and β-chitooligosaccharides from a Loligo opalescens pen with different molecular weights for application in the food industry. The PLA films with native and depolymerized Ch were functionalized through plasma oxygen treatment followed by dip-coating, and their physicochemical properties were assessed by Fourier-transform infrared spectroscopy, X-ray diffraction, water contact angle, and scanning electron microscopy. Their antimicrobial properties were assessed against Escherichia coli and Pseudomonas putida, where Ch-based surfaces reduced the number of biofilm viable, viable but nonculturable, and culturable cells by up to 73%, 74%, and 87%, respectively, compared to PLA. Biofilm growth inhibition was confirmed by confocal laser scanning microscopy. Results suggest that Ch films of higher molecular weight had higher antibiofilm activity under the food storage conditions mimicked in this work, contributing simultaneously to the reuse of marine waste.     

Chitosan composites for bone tissue engineering--an overview

Bone contains considerable amounts of minerals and proteins. Hydroxyapatite [Ca₁₀(PO₄)₆(OH)₂] is one of the most stable forms of calcium phosphate and it occurs in bones as major component (60 to 65%), along with other materials including collagen, chondroitin sulfate, keratin sulfate and lipids. In recent years, significant progress has been made in organ transplantation, surgical reconstruction and the use of artificial protheses to treat the loss or failure of an organ or bone tissue. Chitosan has played a major role in bone tissue engineering over the last two decades, being a natural polymer obtained from chitin, which forms a major component of crustacean exoskeleton. In recent years, considerable attention has been given to chitosan composite materials and their applications in the field of bone tissue engineering due to its minimal foreign body reactions, an intrinsic antibacterial nature, biocompatibility, biodegradability, and the ability to be molded into various geometries and forms such as porous structures, suitable for cell ingrowth and osteoconduction. The composite of chitosan including hydroxyapatite is very popular because of the biodegradability and biocompatibility in nature. Recently, grafted chitosan natural polymer with carbon nanotubes has been incorporated to increase the mechanical strength of these composites. Chitosan composites are thus emerging as potential materials for artificial bone and bone regeneration in tissue engineering. Herein, the preparation, mechanical properties, chemical interactions and in vitro activity of chitosan composites for bone tissue engineering will be discussed.     

Comparative Analysis of the Functional Properties of Films Based on Carrageenans,Chitosan, and Their Polyelectrolyte Complexes

The influence of the structural features of carrageenan on the functional properties of the films was studied. The carrageenans and chitosan films, as well as three-layer films containing a polyelectrolyte complex (PEC) of the two, were prepared. The X-ray diffractograms of carrageenan films reflected its amorphous structure, whereas chitosan and three-layer films were characterized by strong reflection in the regions of 20° and 15° angles, respectively. The SEM of the cross-sectional morphology showed dense packing of the chitosan film, as well as the layer-by-layer structure of different densities for the PEC. Among the tested samples, κ/β-carrageenan and chitosan films showed the highest tensile strength and maximum elongation. Films containing the drug substance echinochrome were obtained. Mucoadhesive properties were assessed as the ability of the films to swell on the mucous tissue and their erosion after contact with the mucosa. All studied films exhibited mucoadhesive properties. All studied films exhibited mucoadhesive properties which depended on the carrageenans structure. Multilayer films are stronger than single-layer carrageenan films due to PEC formation. The resulting puncture strength of the obtained films was comparable to that of commercial samples described in the literature.     

Does the Use of Chitosan Contribute to Oxalate Kidney Stone Formation?

Chitosan is widely used in the biomedical field due its chemical and pharmacological properties. However, intake of chitosan results in renal tissue accumulation of chitosan and promotes an increase in calcium excretion. On the other hand, the effect of chitosan on the formation of calcium oxalate crystals (CaOx) has not been described. In this work, we evaluated the antioxidant capacity of chitosan and its interference in the formation of CaOx crystals in vitro. Here, the chitosan obtained commercially had its identity confirmed by nuclear magnetic resonance and infrared spectroscopy. In several tests, this chitosan showed low or no antioxidant activity. However, it also showed excellent copper-chelating activity. In vitro, chitosan acted as an inducer mainly of monohydrate CaOx crystal formation, which is more prevalent in patients with urolithiasis. We also observed that chitosan modifies the morphology and size of these crystals, as well as changes the surface charge of the crystals, making them even more positive, which can facilitate the interaction of these crystals with renal cells. Chitosan greatly influences the formation of crystals in vitro, and in vivo analyses should be conducted to assess the risk of using chitosan.     

Synthesis and Characterization of 2-Decenoic Acid Modified Chitosan for Infection Prevention and Tissue Engineering

Chitosan nanofiber membranes are recognized as functional antimicrobial materials, as they can effectively provide a barrier that guides tissue growth and supports healing. Methods to stabilize nanofibers in aqueous solutions include acylation with fatty acids. Modification with fatty acids that also have antimicrobial and biofilm-resistant properties may be particularly beneficial in tissue regeneration applications. This study investigated the ability to customize the fatty acid attachment by acyl chlorides to include antimicrobial 2-decenoic acid. Synthesis of 2-decenoyl chloride was followed by acylation of electrospun chitosan membranes in pyridine. Physicochemical properties were characterized through scanning electron microscopy, FTIR, contact angle, and thermogravimetric analysis. The ability of membranes to resist biofilm formation by S. aureus and P. aeruginosa was evaluated by direct inoculation. Cytocompatibility was evaluated by adding membranes to cultures of NIH3T3 fibroblast cells. Acylation with chlorides stabilized nanofibers in aqueous media without significant swelling of fibers and increased hydrophobicity of the membranes. Acyl-modified membranes reduced both S. aureus and P. aeruginosa bacterial biofilm formation on membrane while also supporting fibroblast growth. Acylated chitosan membranes may be useful as wound dressings, guided regeneration scaffolds, local drug delivery, or filtration.     

Customizing Properties of β-Chitin in Squid Pen (Gladius) by Chemical Treatments

The squid pen (gladius) from the Loligo vulgaris was used for preparation of β-chitin materials characterized by different chemical, micro- and nano-structural properties that preserved, almost completely the macrostructural and the mechanical ones. The β-chitin materials obtained by alkaline treatment showed porosity, wettability and swelling that are a function of the duration of the treatment. Microscopic, spectroscopic and synchrotron X-ray diffraction techniques showed that the chemical environment of the N-acetyl groups of the β-chitin chains changes after the thermal alkaline treatment. As a consequence, the crystalline packing of the β-chitin is modified, due to the intercalation of water molecules between β-chitin sheets. Potential applications of these β-chitin materials range from the nanotechnology to the regenerative medicine. The use of gladii, which are waste products of the fishing industry, has also important environmental implications.     

壳聚糖基橡胶树割面喷雾剂及其抗菌防寒效果研究

利用壳聚糖良好的成膜防寒作用和茶树油较强的抗菌杀菌作用,研究一种新型的壳聚糖基橡胶树割面喷雾剂以替代传统的橡胶树割面涂封剂。通过对割面喷雾剂的成膜性能、膜的力学性能、表面结构、耐水性、透气性等性能进行了表征,并研究了割面喷雾剂对条溃疡病主要致病菌柑桔褐腐疫霉的抗菌效果及其对橡胶树割面的防寒效果。实验结果表明,壳聚糖基橡胶树割面喷雾剂具有良好的成膜性能,形成的复合膜不溶于水,拉伸强度σ为5.3 MPa;对CO2的透过率为1.99%,对O2的透过率为13.67%;割面喷雾剂对柑桔褐腐疫霉的抗菌率可达到99.8%;经喷雾剂处理后橡胶树割面的爆皮爆胶寒害现象显著下降,死皮恢复率达90.0%。壳聚糖基橡胶树割面喷雾剂具有良好的抗菌防寒效果,并以喷雾方式代替传统涂封方式,使用方便,可为橡胶树割面保护提供新的技术方法和途径。     

Expression and Characterization of a Novel Cold-Adapted Chitosanase from Marine Renibacterium sp. Suitable for Chitooligosaccharides Preparation

(1) Background: Chitooligosaccharides (COS) have numerous applications due to their excellent properties. Chitosan hydrolysis using chitosanases has been proposed as an advisable method for COS preparation. Although many chitosanases from various sources have been identified, the cold-adapted ones with high stability are still rather rare but required. (2) Methods: A novel chitosanase named CsnY from marine bacterium Renibacterium sp. Y82 was expressed in Escherichia coli, following sequence analysis. Then, the characterizations of recombinant CsnY purified through Ni–NTA affinity chromatography were conducted, including effects of pH and temperature, effects of metal ions and chemicals, and final product analysis. (3) Results: The GH46 family chitosanase CsnY possessed promising thermostability at broad temperature range (0–50 °C), and with optimal activity at 40 °C and pH 6.0, especially showing relatively high activity (over 80% of its maximum activity) at low temperatures (20–30 °C), which demonstrated the cold-adapted property. Common metal ions or chemicals had no obvious effect on CsnY except Mn²⁺ and Co²⁺. Finally, CsnY was determined to be an endo-type chitosanase generating chitodisaccharides and -trisaccharides as main products, whose total concentration reached 56.74 mM within 2 h against 2% (w/v) initial chitosan substrate. (4) Conclusions: The results suggest the cold-adapted CsnY with favorable stability has desirable potential for the industrial production of COS.     

Biochemical Properties and Anti-Biofilm Activity of Chitosan-Immobilized Papain

Chitosan, the product of chitin deacetylation, is an excellent candidate for enzyme immobilization purposes. Here we demonstrate that papain, an endolytic cysteine protease (EC: 3.4.22.2) from Carica papaya latex immobilized on the matrixes of medium molecular (200 kDa) and high molecular (350 kDa) weight chitosans exhibits anti-biofilm activity and increases the antimicrobials efficiency against biofilm-embedded bacteria. Immobilization in glycine buffer (pH 9.0) allowed adsorption up to 30% of the total protein (mg g chitosan⁻¹) and specific activity (U mg protein⁻¹), leading to the preservation of more than 90% of the initial total activity (U mL⁻¹). While optimal pH and temperature of the immobilized papain did not change, the immobilized enzyme exhibited elevated thermal stability and 6–7-fold longer half-life time in comparison with the soluble papain. While one-half of the total enzyme dissociates from both carriers in 24 h, this property could be used for wound-dressing materials design with dosed release of the enzyme to overcome the relatively high cytotoxicity of soluble papain. Our results indicate that both soluble and immobilized papain efficiently destroy biofilms formed by Staphylococcus aureus and Staphylococcus epidermidis. As a consequence, papain, both soluble and immobilized on medium molecular weight chitosan, is capable of potentiating the efficacy of antimicrobials against biofilm-embedded Staphylococci. Thus, papain immobilized on medium molecular weight chitosan appears a presumably beneficial agent for outer wound treatment for biofilms destruction, increasing antimicrobial treatment effectiveness.     

Fish Synucleins: An Update

Synucleins (syns) are a family of proteins involved in several human neurodegenerative diseases and tumors. Since the first syn discovery in the brain of the electric ray Torpedo californica, members of the same family have been identified in all vertebrates and comparative studies have indicated that syn proteins are evolutionary conserved. No counterparts of syns were found in invertebrates suggesting that they are vertebrate-specific proteins. Molecular studies showed that the number of syn members varies among vertebrates. Three genes encode for α-, β- and γ-syn in mammals and birds. However, a variable number of syn genes and encoded proteins is expressed or predicted in fish depending on the species. Among biologically verified sequences, four syn genes were identified in fugu, encoding for α, β and two γ (γ1 and γ2) isoforms, whereas only three genes are expressed in zebrafish, which lacks α-syn gene. The list of “non verified” sequences is much longer and is often found in sequence databases. In this review we provide an overview of published papers and known syn sequences in agnathans and fish that are likely to impact future studies in this field. Indeed, fish models may play a key role in elucidating some of the molecular mechanisms involved in physiological and pathological functions of syn proteins.     

Recent Modifications of Chitosan for Adsorption Applications: A Critical and Systematic Review

Chitosan is considered to be one of the most promising and applicable materials in adsorption applications. The existence of amino and hydroxyl groups in its molecules contributes to many possible adsorption interactions between chitosan and pollutants (dyes, metals, ions, phenols, pharmaceuticals/drugs, pesticides, herbicides, etc.). These functional groups can help in establishing positions for modification. Based on the learning from previously published works in literature, researchers have achieved a modification of chitosan with a number of different functional groups. This work summarizes the published works of the last three years (2012–2014) regarding the modification reactions of chitosans (grafting, cross-linking, etc.) and their application to adsorption of different environmental pollutants (in liquid-phase).     

Purification,Chemical Characterization and Immunomodulatory Activity of a Sulfated Polysaccharide from Marine Brown Algae Durvillaea antarctica

An immunomodulatory polysaccharide (DAP4) was extracted, purified, and characterized from Durvillaea antarctica. The results of chemical and spectroscopic analyses demonstrated that the polysaccharide was a fucoidan, and was mainly composed of (1→3)-α-l-Fucp and (1→4)-α-l-Fucp residues with a small degree of branching at C-3 of (1→4)-α-l-Fucp residues. Sulfate groups were at C-4 of (1→3)-α-l-Fucp, C-2 of (1→4)-α-l-Fucp and minor C-6 of (1→4)-β-d-Galp. Small amounts of xylose and galactose exist in the forms of β-d-Xylp-(1→ and β-d-Gal-(1→. The immunomodulatory activity of DAP4 was measured on RAW 264.7 cells, the results proved that DAP4 exhibited excellent immunomodulatory activities, such as promoted the proliferation of spleen lymphocytes, increased NO production, as well as enhanced phagocytic of macrophages. Besides, DAP4 could also produce better enhancement on the vitality of NK cells. For the high immunomodulatory activity, DAP4 might be a potential source of immunomodulatory fucoidan with a novel structure.     

Foliar application of chitosan activates artemisinin biosynthesis in Artemisia annua L.

There has been much interest in artemisinin owing to its excellent activity against malaria, an infectious disease threatening the tropical world. However, the low artemisinin content (0.01-0.8%, DW) in Artemisia annua, which is the only commercial source of artemisinin, makes artemisinin expensive to produce and not yet available on a global scale. Here we show that foliar application of 100 mg l⁻¹ chitosan improved artemisinin biosynthesis in A. annua. The content of dihydroartemisinic acid and artemisinin in chitosan-treated leaves increased by 72% and 53% compared with control values, respectively. Chitosan induced the expression of ADS and DBR2, which could explain the increase in level of artemisinic metabolites. After chitosan treatment, the amounts of hydrogen peroxide (H₂O₂) and superoxide anion (O₂⁻) in leaves of A. annua were 1.4 and 3.0 times higher than those of the control, respectively. Accumulation of reactive oxygen species (ROS) probably accelerated the conversion of dihydroartemisinic acid to artemisinin. Foliar application of 100 mg l⁻¹ chitosan had no harmful effect on A. annua growth. The simple method described here could be an effective method to improve artemisinin production in A. annua field cultivation.     

The 3/4- and 3/6-Subfamily Variants of α-Conotoxins GI and MI Exhibit Potent Inhibitory Activity against Muscular Nicotinic Acetylcholine Receptors

α-Conotoxins GI and MI belong to the 3/5 subfamily of α-conotoxins and potently inhibit muscular nicotinic acetylcholine receptors (nAChRs). To date, no 3/4- or 3/6-subfamily α-conotoxins have been reported to inhibit muscular nAChRs. In the present study, a series of new 3/4-, 3/6-, and 3/7-subfamily GI and MI variants were synthesized and functionally characterized by modifications of loop2. The results show that the 3/4-subfamily GI variant GI[∆8G]-II and the 3/6-subfamily variants GI[+13A], GI[+13R], and GI[+13K] displayed potent inhibition of muscular nAChRs expressed in Xenopus oocytes, with an IC₅₀ of 45.4–73.4 nM, similar to or slightly lower than that of wild-type GI (42.0 nM). The toxicity of these GI variants in mice appeared to be about a half to a quarter of that of wild-type GI. At the same time, the 3/7-subfamily GI variants showed significantly lower in vitro potency and toxicity. On the other hand, similar to the 3/6-subfamily GI variants, the 3/6-subfamily MI variants MI[+14R] and MI[+14K] were also active after the addition of a basic amino acid, Arg or Lys, in loop2, but the activity was not maintained for the 3/4-subfamily MI variant MI[∆9G]. Interestingly, the disulfide bond connectivity “C1–C4, C2–C3” in the 3/4-subfamily variant GI[∆8G]-II was significantly more potent than the “C1–C3, C2–C4” connectivity found in wild-type GI and MI, suggesting that disulfide bond connectivity is easily affected in the rigid 3/4-subfamily α-conotoxins and that the disulfide bonds significantly impact the variants’ function. This work is the first to demonstrate that 3/4- and 3/6-subfamily α-conotoxins potently inhibit muscular nAChRs, expanding our knowledge of α-conotoxins and providing new motifs for their further modifications.     

Immunostimulative Activity of Low Molecular Weight Chitosans in RAW264.7 Macrophages

Chitosan and its derivatives such as low molecular weight chitosans (LMWCs) have been reported to exert many biological activities, such as antioxidant and antitumor effects. However, complex and molecular weight dependent effects of chitosan remain controversial and the mechanisms that mediate these complex effects are still poorly defined. This study was carried out to investigate the immunostimulative effect of different molecular weight chitosan in RAW264.7 macrophages. Our data suggested that two LMWCs (molecular weight of 3 kDa and 50 kDa) both possessed immunostimulative activity, which was dependent on dose and, at the higher doses, also on the molecular weight. LMWCs could significantly enhance the the pinocytic activity, and induce the production of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), interferon-γ (IFN-γ), nitric oxide (NO) and inducible nitric oxide synthase (iNOS) in a molecular weight and concentration-dependent manner. LMWCs were further showed to promote the expression of the genes including iNOS, TNF-α. Taken together, our findings suggested that LMWCs elicited significantly immunomodulatory response through up-regulating mRNA expression of proinflammatory cytokines and activated RAW264.7 macrophage in a molecular weight- and concentration-dependent manner.     

A New Lyngbyatoxin from the Hawaiian Cyanobacterium Moorea producens

Lyngbyatoxin A from the marine cyanobacterium Moorea producens (formerly Lyngbya majuscula) is known as the causative agent of “swimmer’s itch” with its highly inflammatory effect. A new toxic compound was isolated along with lyngbyatoxin A from an ethyl acetate extract of M. producens collected from Hawaii. Analyses of HR-ESI-MS and NMR spectroscopies revealed the isolated compound had the same planar structure with that of lyngbyatoxin A. The results of optical rotation and CD spectra indicated that the compound was a new lyngbyatoxin A derivative, 12-epi-lyngbyatoxin A (1). While 12-epi-lyngbyatoxin A showed comparable toxicities with lyngbyatoxin A in cytotoxicity and crustacean lethality tests, it showed more than 100 times lower affinity for protein kinase Cδ (PKCδ) using the PKCδ-C1B peptide when compared to lyngbyatoxin A.