Activity of a Recombinant Chitinase of the Atta sexdens Ant on Different Forms of Chitin and Its Fungicidal Effect against Lasiodiplodia theobromae.

This study evaluates the activity of a recombinant chitinase from the leaf-cutting ant Atta sexdens (AsChtII-C4B1) against colloidal and solid α- and ß-chitin substrates. 1H NMR analyses of the reaction media showed the formation of N-acetylglucosamine (GlcNAc) as the hydrolysis product. Viscometry analyses revealed a reduction in the viscosity of chitin solutions, indicating that the enzyme decreases their molecular masses. Both solid state 13C NMR and XRD analyses showed minor differences in chitin crystallinity pre- and post-reaction, indicative of partial hydrolysis under the studied conditions, resulting in the formation of GlcNAc and a reduction in molecular mass. However, the enzyme was unable to completely degrade the chitin samples, as they retained most of their solid-state structure. It was also observed that the enzyme acts progressively and with a greater activity on α-chitin than on ß-chitin. AsChtII-C4B1 significantly changed the hyphae of the phytopathogenic fungus Lasiodiplodia theobromae, hindering its growth in both solid and liquid media and reducing its dry biomass by approximately 61%. The results demonstrate that AsChtII-C4B1 could be applied as an agent for the bioproduction of chitin derivatives and as a potential antifungal agent.

Multifunctional bilayer membranes composed of poly(lactic acid), beta-chitin whiskers and silver nanoparticles for wound dressing applications.

Nanomaterial-based wound dressings have been extensively studied for the treatment of both minor and life-threatening tissue injuries. These wound dressings must possess several crucial characteristics, such as tissue compatibility, non-toxicity, appropriate biodegradability to facilitate wound healing, effective antibacterial activity to prevent infection, and adequate physical and mechanical strength to withstand repetitive dynamic forces that could potentially disrupt the healing process. Nevertheless, the development of nanostructured wound dressings that incorporate various functional micro- and nanomaterials in distinct architectures, each serving specific purposes, presents significant challenges. In this study, we successfully developed a novel multifunctional wound dressing based on poly(lactic acid) (PLA) fibrous membranes produced by solution-blow spinning (SBS) and electrospinning. The PLA-based membranes underwent surface modifications aimed at tailoring their properties for utilization as effective wound dressing platforms. Initially, beta-chitin whiskers were deposited onto the membrane surface through filtration, imparting hydrophilic character. Afterward, silver nanoparticles (AgNPs) were incorporated onto the beta-chitin layer using a spray deposition method, resulting in platforms with antimicrobial properties against both Staphylococcus aureus and Escherichia coli. Cytotoxicity studies demonstrated the biocompatibility of the membranes with the neonatal human dermal fibroblast (HDFn) cell line. Moreover, bilayer membranes exhibited a high surface area and porosity (> 80%), remarkable stability in aqueous media, and favorable mechanical properties, making them promising candidates for application as multifunctional wound dressings.

N-(2-hydroxy)-propyl-3-trimethylammonium, O-palmitoyl chitosan: Synthesis, physicochemical and biological properties.

Two samples of N-(2-hydroxy)-propyl-3-trimethylammonium, O-palmitoyl chitosan (DPCat) with different average degrees of quaternization named as DPCat35 (DQ¯ = 35%) and DPCat80 (DQ¯ = 80%), were successfully synthesized by reacting glycidyltrimethylammonium chloride (GTMAC) with O-palmitoyl chitosan (DPCh) derivative (DS¯ = 12%). Such amphiphilic derivatives of chitosan were fully water-soluble at 1.0 < pH < 12.0 and showed significant electrostatic stability enhancement of a self-assembly micellar nanostructure (100-320 nm) due to its positively-charged out-layer. In vitro mucoadhesive and cytotoxicity essays toward healthy fibroblast cells (Balb/C 3T3 clone A31 cell), human prostate cancer (DU145) and liver cancer (HepG2/C3A) cell lines revealed that the biological properties of DPCat derivatives were strongly dependent on DQ¯. Additionally, DPCat35 had better interactions with the biological tissue and with mucin glycoproteins at pH 7.4 as well as exhibited potential to be used on the development of drug delivery systems for prostate and liver cancer treatment.

Tuning the properties of carboxymethylchitosan-based porous membranes for potential application as wound dressing.

Wound repair is a complex process that calls for strategies to allow a rapid and effective regeneration of injured skin, which has stimulated the research of advanced wound dressings. Herein, highly porous membranes of N,O-carboxymethylchitosan (CMCh), and poly (vinyl alcohol) (PVA) were successfully prepared via a green and facile freeze-drying method of blend solutions containing CMCh/PVA at weight ratio 25/75. Membranes composed only by CMCh were also prepared and genipin was used for crosslinking. Different contents of TiO2 nanoparticles were incorporated to both type of membranes, which were characterized in terms of morphology, porosity (Φ), swelling capacity (S.C.), mechanical properties, susceptibility to lysozyme degradation and in vitro cytotoxicity toward human fibroblast (HDFn) and keratinocytes (HaCaT) cells. Larger apparent pores were observed in the surface of the genipin-crosslinked CMCh membrane, which resulted in higher porosity (Φ ≈ 76%) and swelling capacity (S.C. ≈ 1720%) as compared to CMCh/PVA membrane (Φ ≈ 68%; S.C. ≈ 1660%). The porosity of both types of membranes decreased upon the addition of TiO2 nanoparticles while swelling capacity increased. Due to their high porosity and swelling capacity, adequate mechanical properties, controlled degradability, and cytocompatibility, such carboxymethylchitosan-based membranes are potentially useful as wound dressings.

Tracking Sulfonated Polystyrene Diffusion in a Chitosan/Carboxymethyl Cellulose Layer-by-Layer Film: Exploring the Internal Architecture of Nanocoatings.

Since chitosan presents the ability to interact with a wide range of molecules, it has been one of the most popular natural polymers for the construction of layer-by-layer thin films. In this study, depth-profiling X-ray photoelectron spectroscopy (XPS) was employed to track the diffusion of sulfonated polystyrene (SPS) in carboxymethyl cellulose/chitosan (CMC/Chi) multilayers. Our findings suggest that the CMC/Chi film does not constitute an electrostatic barrier sufficient to block diffusion of SPS, and that diffusion can be controlled by adjusting the diffusion time and the molecular weight of the polymers that compose the CMC/Chi system. In addition to monitoring the diffusion, it was also possible to observe a process of preferential interaction between Chi and SPS. Thus, the nitrogen N 1s peak, due to functional groups found exclusively in chitosan chains, was the key factor to identifying the molecular interactions involving chitosan and the different polyanions. Accordingly, the presence of a strong polyanion such as SPS shifts the N 1s peak to a higher level of binding energy. Such results highlight that understanding the fundamentals of polymer interactions is a major step to fine-tuning the internal architecture of LbL structures for specific applications (e.g., drug release).

Bioproduction of N-acetyl-glucosamine from colloidal α-chitin using an enzyme cocktail produced by Aeromonas caviae CHZ306.

N-acetyl-D-glucosamine (GlcNAc) is an important amino-monosaccharide with great potential for biotechnological applications. It has traditionally been produced by the chemical hydrolysis of chitin, despite certain industrial and environmental drawbacks, including acidic wastes, low yields and high costs. Therefore, enzymatic production has gained attention as a promising environmentally-friendly alternative to the chemical processes. In this study we demonstrate the GlcNAc bioproduction from colloidal α-chitin using an enzyme cocktail containing endochitinases and exochitinases (chitobiosidases and N-acetyl-glucosaminidases). The enzyme cocktail was extracted after fermentation in a bioreactor by Aeromonas caviae CHZ306, a chitinolytic marine bacterium with great potential for chitinase production. Hydrolysis parameters were studied in terms of temperature, pH, enzyme and substrate concentration, and reaction time, achieving over 90% GlcNAc yield within 6 h. The use of colloidal α-chitin as substrate showed a substantial improvement of GlcNAc yields, when compared with ß-chitin and α-chitin polymorphs. Such result is directly related to a significant decrease in crystallinity and viscosity from natural α-chitin, providing the chitinase with greater accessibility to the depolymerized chains. This study provides valuable information on the GlcNAc bioproduction from chitin using an enzymatic approach, addressing the key points for its production, including the enzyme cocktail composition and the substrate structures.

Chitosan microparticles embedded with multi-responsive poly(N-vinylcaprolactam-co-itaconic acid-co-ethylene-glycol dimethacrylate)-based hydrogel nanoparticles as a new carrier for delivery of hydrophobic drugs.

In this paper, chitosan was used as protective agent for dual temperature-/pH-sensitive poly(N-vinylcaprolactam-co-itaconic acid-co-ethylene- glycol dimethacrylate)- based hydrogel nanoparticles (poly(NVCL-co-IA-co-EGDMA)) aiming avoid their undesirable colloidal destabilization at different conditions of body human tissues. Thus, poly(NVCL-co-IA-co-EGDMA) was embedded into chitosan and a new solid dispersion was prepared via spray-drying and ketoprofen was used as carrier. Two different sizes of hydrogel nanoparticles (120.6 nm and 185.9 nm) were evaluated and they exhibited a drug encapsulation efficiency of the 39.6% and 57.8%, respectively. The smaller nanoparticles showed to be faster for releasing of ketoprofen at pH 7.4 and 37 °C due to their larger surface area and higher swelling ability. Chitosan played a role of a secondary barrier for the ketoprofen diffusion, extending its release compared to hydrogel nanoparticles alone. Among two concentrations (40 wt% and 70 wt%) of hydrogel nanoparticles related to chitosan, the first one induced higher percentages of ketoprofen release: 74.2% against 64.6%. In addition, the interactions between chitosan matrix and poly(NVCL-co-IA-co-EGDMA) did not change the multi-responsive behavior of hydrogels, suggesting the chitosan was efficient for keeping integrity of nanoparticles hydrogels. Chitosan/poly(NVCL-co-IA-co-EGDMA) hybrid microparticles seems to be a promising new carrier for release of hydrophobic drugs, such as ketoprofen.

Characterization and physical-chemistry of methoxypoly(ethylene glycol)-g-chitosan.

Methoxypoly(ethyleneglycol)-graft-chitosan (PEG-g-Ch) was prepared by grafting polyethyleneglycol into chitosans (Ch) exhibiting different average degree of deacetylation (60% < DD¯â€¯< 95%). 1H NMR showed that PEG-g-Ch derivatives presented high average degree of N-substitution (DS¯â€¯≈ 40%) and such derivatives exhibited full water solubility at 1.0 < pH < 11.0. The mPEG-g-Ch derivatives displayed much lower intrinsic viscosity (20 mL g-1 < [η] < 110 mL g-1) as compared to the parent chitosans (440 mL g-1 < [η] < 1650 mL g-1) due to extensive exposition of PEG chains to the aqueous medium and compact coiling of the chitosan backbone. The presence of numerous PEG chains grafted into chitosan also determined the crystalline arrangement and the thermal stability of PEG-g-Ch derivatives. The rheological study showed that the concentrated aqueous solutions of PEG-g-Ch derivatives displayed pseudoplastic behavior regardless of the parent chitosans´ characteristics and no dependence of dynamic viscosity on the temperature. However, PChD2 (DD¯â€¯≈ 76%; [η] ≈ 1201 mL g-1) showed a distinct rheological behavior as it formed a physically cross-linked hydrogel that exhibited a thermo-induced sol-gel transition at ≈38 °C.

Gellan Gum/Pectin Beads Are Safe and Efficient for the Targeted Colonic Delivery of Resveratrol.

This work addresses the establishment and characterization of gellan gum:pectin (GG:P) biodegradable mucoadhesive beads intended for the colon-targeted delivery of resveratrol (RES). The impact of the polymer carrier system on the cytotoxicity and permeability of RES was evaluated. Beads of circular shape (circularity index of 0.81) with an average diameter of 914 µm, Span index of 0.29, and RES entrapment efficiency of 76% were developed. In vitro drug release demonstrated that beads were able to reduce release rates in gastric media and control release for up to 48 h at an intestinal pH of 6.8. Weibull's model correlated better with release data and b parameter (0.79) indicated that the release process was driven by a combination of Fickian diffusion and Case II transport, indicating that both diffusion and swelling/polymer chains relaxation are processes that contribute equally to control drug release rates. Beads and isolated polymers were observed to be safe for Caco-2 and HT29-MTX intestinal cell lines. RES encapsulation into the beads allowed for an expressive reduction of drug permeation in an in vitro triple intestinal model. This feature, associated with low RES release rates in acidic media, can favor targeted drug delivery from the beads in the colon, a promising behavior to improve the local activity of RES.

Structure, morphology and properties of genipin-crosslinked carboxymethylchitosan porous membranes.

Highly porous genipin cross-linked membranes of carboxymethylchitosan exhibiting different crosslinking degree (3%

Chitosan-based biomaterials used in critical-size bone defects: radiographic study in rat's calvaria / Biomateriais a base de quitosana na correção de defeitos ósseos críticos criados em calvaria de ratos: avaliação radiográfica

Objetivo: Este estudo avaliou através de imagens radiográficas digitais, a ação de biomateriais de quitosana e de cloridrato de quitosana, com baixo e alto peso molecular, utilizados na correção de defeitos ósseos de tamanho crítico (DOTC)em calvária de ratos. Material e MétodoO: DOTCs com 8 mm de diâmetro foram criados cirurgicamente na calvária de 50 ratos Holtzman. Em 10 animais o defeito foi preenchido foram preenchidos com coágulo sanguíneo (controle negativo). Os 40 animais restantes foram divididos de acordo com o biomaterial utilizado no preenchimento do defeito (quitosana de baixo peso e de alto peso molecular, e cloridrato de quitosana de baixo e de alto peso molecular), e foram avaliados em dois períodos experimentais (15 e 60 dias), totalizando 5 animais/biomaterial/período de avaliação. Resultado: A avaliação radiográfica foi feita utilizando duas radiografias digitais do crânio do animal: uma tomada logo após o defeito ósseo ser criado e a outra no momento do sacrifício. Nessas imagens, foi avaliada a densidade óssea radiográfica inicial e a final na área do defeito, que foram comparadas. As análises na densidade óssea radiográfica indicaram aumento da densidade óssea radiográfica dos DOTCs tratados para todos os biomateriais testados, em ambos os períodos. Resultados semelhantes foram encontrados no grupo controle. Conclusão: Conclui-se que os biomateriais de quitosana testados não foram capazes de aumentar a densidade radiográfica em DOTC realizados em calvária de ratos.

Objective: This study evaluated, using digital radiographic images, the action of chitosan and chitosan hydrochloride biomaterials, with both low and high molecular weight, used in the correction of critical-size bone defects (CSBD's) in rat's calvaria. Material and Method: CSBD's with 8 mm in diameter were surgically created in the calvaria of 50 Holtzman rats and these were filled with a blood clot (Control), low molecular weight chitosan, high molecular weight chitosan, low molecular weight chitosan hydrochloride and high molecular weight chitosan hydrochloride, for a total of 10 animals, which were divided into two experimental periods (15 and 60 days), for each biomaterial. The radiographic evaluation was made using two digital radiographs of the animal's skull: one taken right after the bone defect was created and the other at the moment of the sacrifice, providing the initial and the final radiographic bone density in the area of the defect, which were compared. Result: Analysis of radiographic bone density indicated that the increase in the radiographic bone density of the CSBD's treated with the proposed biomaterials, in either molecular weight, in both observed periods, where similar to those found in control group. Conclusion: Tested chitosan-based biomaterials were not able to enhance the radiographic density in the CSBD's made in rat's calvaria.

Different Molecular Weight Chitosan-Based Membranes for Tissue Regeneration.

Natural polymers, such as chitosan, obtained from chitin, are been widely studied for use in the tissue regeneration field. This study established a protocol to attain membranes made from this biopolymer, consisting of high or low molecular weight chitosan. The biocompatibility of these membranes was histologically evaluated, comparing them to collagen membrane surgically implanted in rat subcutaneous tissue. Fifteen Holtzmann rats were divided in three experimental groups: High and Low Molecular Weight Chitosan membranes (HMWC and LMWC) and Collagen membranes (C-control group); each of them with three experimental periods: 7, 15 and 30 days. As a result, after the seven days evaluation, the membranes were present and associated with a variable degree of inflammation, and after the 15 and 30 days evaluations, the membranes were absent in all groups. It is concluded that the chitosan-based membranes were successfully attained and presented comparable resorption times to collagen membranes.

Histological evaluation of chitosan-based biomaterials used for the correction of critical size defects in rat's calvaria.

Chitosan, a biopolymer obtained from chitin, and its derivates, such as chitosan hydrochloride, has been reported as wound healing accelerators and as possible bone substitutes for tissue engineering, and therefore these substances could be relevant in dentistry and periodontology. The purpose of this investigation was to make a histological evaluation of chitosan and chitosan hydrochloride biomaterials (gels) used in the correction of critical size bone defects made in rat's calvaria. Bone defects of 8 mm in diameter were surgically created in the calvaria of 50 Holtzman (Rattus norvegicus) rats and filled with blood clot (control), low molecular weight chitosan, high molecular weight chitosan, low molecular weight chitosan hydrochloride, and high molecular weight chitosan hydrochloride, numbering 10 animals, divided into two experimental periods (15 and 60 days), for each biomaterial. The histological evaluation was made based on the morphology of the new-formed tissues in defect's region, and the results indicated that there was no statistical difference between the groups when the new bone formation in the entire defect's area were compared (p > 0.05) and, except in the control groups, assorted degrees of inflammation could be seen. In conclusion, chitosan and chitosan hydrochloride biomaterials used in this study were not able to promote new bone formation in critical size defects made in rat's calvaria.

Prevenção de aderências pericárdicas pós-operatórias com uso de carboximetilquitosana termoestéril / Prevention of postoperative pericardial adhesions using thermal sterile carboxymethyl chitosan

OBJETIVO: Este trabalho tem como objetivo avaliar alterações físico-químicas da carboximetilquitosana após termoesterilização e sua eficácia na prevenção de aderências pericárdicas pós-esternotomia. MÉTODOS: Após ser submetida a termoesterilização em autoclave, a carboximetilquitosana termoestéril (CMQte) foi submetida a análises físico-químicas. Doze animais foram divididos em dois grupos e submetidos à pericardiotomia e a protocolo de indução de aderências. A seguir, foi aplicada de forma tópica a CMQte ou solução salina. Após 8 semanas, foi realizada esternotomia e avaliação macroscópica do grau de aderências, tempo de dissecção e quantidade do uso de dissecção cruenta e avaliação microscópica. RESULTADOS: As análises físico-químicas não mostraram diferença entre a CMQ e CMQte. A avaliação macroscópica mostrou que a intensidade das aderências foi significantemente menor no grupo CMQte (P=0,007). O tempo de dissecção e o uso de dissecção cruenta também apresentaram reduções significativas (P=0,007, P=0,008; respectivamente). CONCLUSÃO: O método de esterilização empregado não alterou as propriedades físico-químicas da carboximetilquitosana. O uso de biopolímeros de barreira como a CMQte pode reduzir a intensidade das aderências pós-cirúrgicas no pericárdio, diminuindo as complicações da esternotomia em reoperações cardiovasculares.

OBJECTIVE: The aim of this study is to evaluate CMC physical-chemical alterations after thermal sterilization and its efficacy in preventing poststernotomy pericardial adhesions. METHODS: After autoclaving thermal sterilization, thermal sterile Carboxymethyl Chitosan (CMCts) was submitted to physical-chemical analysis. Twelve animals were divided into two groups and underwent pericardiotomy and adhesion induction protocol. Afterward, topic CMCts or saline solution was administered. After 8 weeks, a sternotomy was performed for adhesion score macroscopic evaluation, dissection time and the amount of recalcitrant dissection, and microscopic evaluation. RESULTS: Physical-chemical analysis showed no difference between CMC and CMCts. A macroscopic analysis showed that the intensity of adhesions was significantly lower in the CMCts group (P=0.007). Dissection time and use of recalcitrant dissection also decreased significantly (P=0.007, P=0.008; respectively). Microscopic results indicated a significant reduction in the epicardium collagen area and in the total epicardium area (P=0.05) and (P=0.03). CONCLUSION: The sterilization method did not change Carboxymethyl Chitosan physical-chemical properties. Using barrier bipolymer, such as CMCts, can decrease the intensity of pericardium postoperative adhesions, reducing sternotomy complications in cardiovascular reoperations.

Prevention of postoperative pericardial adhesions using thermal sterile carboxymethyl chitosan.

OBJECTIVE: The aim of this study is to evaluate CMC physical-chemical alterations after thermal sterilization and its efficacy in preventing poststernotomy pericardial adhesions. METHODS: After autoclaving thermal sterilization, thermal sterile Carboxymethyl Chitosan (CMCts) was submitted to physical-chemical analysis. Twelve animals were divided into two groups and underwent pericardiotomy and adhesion induction protocol. Afterward, topic CMCts or saline solution was administered. After 8 weeks, a sternotomy was performed for adhesion score macroscopic evaluation, dissection time and the amount of recalcitrant dissection, and microscopic evaluation. RESULTS: Physical-chemical analysis showed no difference between CMC and CMCts. A macroscopic analysis showed that the intensity of adhesions was significantly lower in the CMCts group (P=0.007). Dissection time and use of recalcitrant dissection also decreased significantly (P=0.007, P=0.008; respectively). Microscopic results indicated a significant reduction in the epicardium collagen area and in the total epicardium area (P=0.05) and (P=0.03). CONCLUSION: The sterilization method did not change Carboxymethyl Chitosan physical-chemical properties. Using barrier biopolymer, such as CMCts, can decrease the intensity of pericardium postoperative adhesions, reducing sternotomy complications in cardiovascular reoperations.