Dibutyryl Chitin Research Articles

Fabrication of chitin monoliths with controllable morphology by thermally induced phase separation of chemically modified chitin

As a natural polymer, chitin has excellent biological properties such as biodegradability and immunological, antibacterial, and wound-healing activities and has numerous applications in cosmetics, drug delivery, and pharmaceuticals. Organic polymer monoliths have also drawn significant attention, owing to their high permeability, large surface area, and high mechanical strength. They are usually applied to separation, ion exchange, catalysis, and chromatography. We have previously prepared cellulose monoliths using biopolymers; however, because chitin possesses amide groups on its side chain, it is superior to cellulose for further chemical modification and applications. However, the utilization of chitin is restricted by its insolubility in water and common organic solvents. In this study, for the first time, a monolith was prepared by chemical modification of chitin using a thermally induced phase separation (TIPS) method. First, we prepared dibutyrylchitin (DBC) as a starting polymer that is soluble in organic solvents. To prepare the monolith, DBC was dissolved completely in dimethyl sulfoxide (DMSO) while heating, and deionized water was added to the solution. It was then cooled at 20 °C to form a monolith via phase separation. The porous morphology of the DBC monolith was altered by regulating the DBC concentration, DMSO/H2O ratio, and aging temperature. The DBC monolith was converted to a chitin monolith by the alkaline hydrolysis of butyryl ester. The successful hydrolysis of butyryl ester was confirmed by the disappearance of the peak at 1735 cm−1 in the FT-IR spectra, which is related to the ester moiety of DBC. The chitin monolith has the potential to be utilized under water flow for catalysis, metal capture from wastewater, dye sorption, and drug delivery systems.

A New Concept in Minimally Invasive Embryo Transfer

Abstract Considerable variation in embryo transfer (ET) catheter types, diverging opinions on their quality and functionality, complications following the insertion of catheters, low efficiency of the application of ET methods in humans, and their widely varying efficiency in animals demonstrate the need to improve ET methods and to look for new types of catheters. Such an opportunity is offered by the introduction of catheters made of new-generation biomaterials. This study was aimed to introduce a new generation of biomaterials into reproductive biotechnology. New-generation materials were compared with materials that have been used for many years, and the functionality of newly produced catheters was compared in vivo. Five types of biomaterials were tested: polycaprolactone (PCL), dibutyryl chitin (DBC), polypropylene (PP), polyethylene (PE) and polylactide (PLA). The study was carried out in two stages. Firstly, the basic utility parameters such as geometric stability, surface structure and catheter resistance were evaluated. Subsequently, the biocompatibility of selected biomaterials in embryo cultures was examined, and the development potential of the obtained blastocysts was evaluated. In the second stage, in vivo with live animals, the biomaterials were tested for biocompatibility and the obtained catheters were examined for their ET functionality. Efficiency with the use of the newly produced catheters was determined, the quality of the blastocysts obtained after embryo culture in the uterus was assessed, and oviducts were subjected to histopathological examination after embryo transfer. Of the tested biomaterials, only polyethylene (PE) showed adequate biological and material properties and proved suitable for production of ET catheters.

Chitin and Chitinases: Biomedical And Environmental Applications of Chitin and its Derivatives

Disposal of chitin wastes from crustacean shell can cause environmental and health hazards. Chitin is a well known abundant natural polymer extracted after deproteinization and demineralization of the shell wastes of shrimp, crab, lobster, and krill. Extraction of chitin and its derivatives from waste material is one of the alternative ways to turn the waste into useful products. Chitinases are enzymes that degrade chitin. Chitinases contribute to the generation of carbon and nitrogen in the ecosystem. Chitin and chitinolytic enzymes are gaining importance for their biotechnological applications. The presence of surface charge and multiple functional groups make chitin as a beneficial natural polymer. Due to the reactive functional groups chitin can be used for the preparation of a spectrum of chitin derivatives such as chitosan, alkyl chitin, sulfated chitin, dibutyryl chitin and carboxymethyl chitin for specific applications in different areas. The present review is aimed to summarize the efficacy of the chitinases on the chitin and its derivatives and their diverse applications in biomedical and environmental field. Further this review also discusses the synthesis of various chitin derivatives in detail and brings out the importance of chitin and its derivatives in biomedical and environmental applications.

Effects of electrospun scaffolds of di-O-butyrylchitin and poly-(ε-caprolactone) on wound healing.

We sought to determine the usefulness of electrospun dibutyrylchitin (DBC) or poly-(ε-caprolactone [PCL]), in wound treatment. We investigated the mechanisms of action of these polymers on wound healing. We synthesized DBC, a newly identified ester derivative of chitin, using a patented method comprising the substitution of butyryl groups at positions C-3 and C-6 in chitin molecules. We confirmed the double substitution by the butyric groups using infrared spectrometry. The fibrous scaffolds were obtained using the electrospinning method. A polypropylene net was implanted subcutaneously in the rat and served as a wound model. Both DBC and PCL increased granulation tissue weight in the wound. In contrast to PCL, DBC did not abolish glycosaminoglycan changes in wounds. The tested samples did not impair total collagen synthesis or induce excessive fibrosis. In both PCL- and DBC-treated wounds, we observed a lower level of soluble collagen (compared with controls). The results show better hydration of the wounds in both the DBC and PCL groups. No induction of large edema formation by the tested materials was observed. These polymers induced almost identical macrophage-mediated reactions to foreign-body implantation. The implants increased the blood vessel number in a wound. Both PCL and DBC could be used as scaffolds or dressings for wound treatment. The materials were safe and well tolerated by animals. As DBC did not disturb glycosaminoglycan accumulation in wounds and absorbed twice as much liquid as PCL, it can be considered superior.

Synthesis and physicochemical characterization of chitin dihexanoate — A new biocompatible chitin derivative — In comparison to chitin dibutyrate

Chitin dihexanoate (DHCH) is the novel biocompatible and technologically friendly highly substituted chitin diester. Here we described optimization of DHCH and chitin dibutyrate (dibutyryl chitin, DBC) synthesis conditions (temperature and reaction time) to obtain desired polymers with high reaction yield, high substitution degree (close to 2) and appropriately high molecular weights. A two-step procedure, employing acidic anhydrides (hexanoic or butyric anhydride) as the acylation agent and methanesulfonic acid both as the catalyst and the reaction medium, was applied. Chemical structures of DBC and DHCH were confirmed by NMR (1H and 13C) and IR investigations. Mechanical properties, thermogravimetric analysis, differential scanning calorimetry and biocompatibility (Neutral red uptake assay, Skin Sensitization and Irritation Tests) were assessed. Both polymers proved highly biocompatible (non-cytotoxic in vitro, non-irritating and non-allergic to skin) and soluble in several organic solvents (dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetone, ethanol and others). It is worth emphasizing that DHCH and DBC can be easily processed by solvent casting method and the salt-leaching method, what gives the opportunity to prepare highly porous structures, which can be further successfully applied as the material for wound dressings and scaffolds for tissue engineering.

Fibrous Polymeric Composites Based on Alginate Fibres and Fibres Made of Poly-ε-caprolactone and Dibutyryl Chitin for Use in Regenerative Medicine

This work concerns the production of fibrous composite materials based on biodegradable polymers such as alginate, dibutyryl chitin (DBC) and poly-ε-caprolactone (PCL). For the production of fibres from these polymers, various spinning methods were used in order to obtain composite materials of different composition and structure. In the case of alginate fibres containing the nanoadditive tricalcium phosphate (TCP), the traditional method of forming fibres wet from solution was used. However in the case of the other two polymers the electrospinning method was used. Two model systems were tested for biocompatibility. The physicochemical and basic biological tests carried out show that the submicron fibres produced using PCL and DBC have good biocompatibility. The proposed hybrid systems composed of micrometric fibres (zinc and calcium alginates containing TCP) and submicron fibres (DBC and PCL) meet the requirements of regenerative medicine. The biomimetic fibre system, the presence of TCP nanoadditive, and the use of polymers with different resorption times provide a framework with specific properties on which bone cells are able to settle and proliferate.

Inhibitory effect of dibutyryl chitin ester on nitric oxide and prostaglandin E2 production in LPS-stimulated RAW 264.7 cells

Inflammation is a highly complex process that protects against foreign challenge or tissue injury. The ester derivative dibutyryl chitin (DBC) reportedly accelerates wound healing and exerts an anti-inflammatory effect. However, little is known regarding the inhibitory effect of DBC in anti-inflammation. In this study, we investigated the effect of DBC on the inducible nitric oxide synthetase (iNOS) and cyclooxygenage-2 (COX-2) pathways and pro-inflammatory cytokine production in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. Our results demonstrate that DBC (MW 3,772) significantly inhibits overproduction of NO and PGE(2) as well as pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-1β, in LPS-stimulated RAW 264.7 macrophages. Inhibition of NO and PGE(2) overproduction in LPSstimulated RAW 264.7 macrophages by DBC was mediated through the down-regulation of iNOS and COX-2 expression. These results demonstrate that DBC efficiently inhibits inflammation and has potential as an effective anti-inflammatory and wound healing agent.

Effect of Electrospun Non-Woven Mats of Dibutyryl Chitin/Poly(Lactic Acid) Blends on Wound Healing in Hairless Mice

The aim of this study was to examine the proliferative ability of dibutyryl chitin (DBC) on scratch wounds in HaCaT keratinocytes and to evaluate the effect of nanoporous non-woven mat (DBCNFM) on skin wound healing in hairless mice using the advantages of DBCNFM, such as high porosity and high surface area to volume. The cell spreading activity of DBC was verified through a cell spreading assay in scratched human HaCaT keratinocytes. Scratch wound experiments showed that DBC notably accelerates the spreading rate of HaCaT keratinocytes in a dose dependent manner. The molecular aspects of the healing process were also investigated by hematoxylin & eosin staining of the healed skin, displaying the degrees of reepithelialization and immunostaining on extracellular matrix synthesis and remodeling of the skin. Topical application of DBCNFM significantly reduced skin wound rank scores and increased the skin remodeling of the wounded hairless mice in a dose dependent way. Furthermore, DBCNFM notably increased the expression of the type 1 collagen and filaggrin. These results demonstrate that DBC efficiently accelerates the proliferation of HaCaT keratinocytes and DBCNFM notably increases extracellular matrix synthesis on remodeling of the skin, and these materials are a good candidate for further evaluation as an effective wound healing agent.

Evaluation of dibutyrylchitin as new excipient for sustained drug release

Dibutyrylchitin (DBC), a lipophilic chitin diester, has been synthesized from chitin and butyric anhydride with methanesulfonic acid as catalyst. Exhaustive esterification of free alcoholic groups of chitin was assessed by FT-IR and 1H-NMR spectroscopy. High degree of alkyl substitution allowed DBC to acquire an almost completely lipophilic character. Tablets of paracetamol and metformin employing DBC as major excipient, in comparison with starch, microcrystalline cellulose, lactose and polyvinylpyrrolidone, were prepared and rates of drug release were checked by dissolution test assays. DBC released drug at a lower rate than that of the other tested materials. A comparison study of rate release of metformin from DBC tablets and from metformin-hydroxypropyl methylcellulose prolonged release oral formulation available on the market has been also curried out. Under the same conditions and in the presence of the same amount of loaded drug, DBC released 64% of metformin whereas hypromellose-based tablets released 87%.

The impact of the dibutyrylchitin molar mass on the bioactive properties of dressings used to treat soft tissue wounds

In this work, we describe a novel technique for producing biocompatible medical products with bioactive properties from the biodegradable polymer dibutyrylchitin (DBC). Materials produced by blowing out polymer solutions have excellent hemostaic properties and are easy to handle during surgery. Biocompatibility studies, encompassing hemostasis and the evaluation of post-implantation reactions, indicate that the biological properties of DBC depend on the molecular mass of the polymer. Lower molecular mass polymers are preferable for use in implanted wound dressings.

The release of active substances from selected carbohydrate biopolymer membranes

Abstract As a biopolymer application to control release systems is increasing at present, flat matrices (transdermal systems) should be highlighted. They constitute one of the most friendly form of drug administration to the patient. The present results concern investigations on the active substance release (ibuprofen and salicylic acid) from film matrices made from biopolymers: polylactid acid (PLA), dibutyrylchitin (DBC) and chitosan (CH). The amount of released active substance was examined with UV-VIS spectrophotometer. The release process was conducted in the medium of pH = 5.6 as the transdermal systems are applied to human skin surface of pH value approximating 5.6. Swelling of polymer samples was confirmed in the buffer of pH = 5.6 as well. The paper comprises the analysis of obtained release results according to the proposed two stage complex diffusion model.

Synthesis and solubility properties of chitin acetate/butyrate copolymers

AbstractIn this article, syntheses of chitin acetate/butyrate copolymers with different contents of acetyl residue in polymer chain have been described. Chitin copolymers as well as pure diacetylchitin (DAC) and dibutyrylchitin (DBC) have been obtained in the reaction of shrimp chitin with mixture of acetic and butyric anhydrides used in different proportion. Reactions have been carried out under heterogeneous condition with perchloric acid (PA) used as a catalyst of reaction. Chemical structure of DAC, DBC, and co‐(acetate/butyrates) was confirmed in 1H‐NMR as well as in Fourier transform infrared (FTIR) investigations. Results of 1H‐NMR investigation showed that in all cases of chitin esterification carried out at applied conditions, final products with summary degree of substitution (DP) closed to 2 have been obtained. Prepared chitin acetate/butyrate copolymers with high degree of polymerization (DP) (based on value of intrinsic viscosity) with varying amount of acetyl groups from 0.24 to 1.29 have been formed depending on the mixture composition of acetic and butyric acid anhydrides. All obtained co‐(acetate/butyrates) are soluble in organic solvents such as dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide (DMAc) as well as ethanol and form strong transparent films. It was stated that solubility of obtained chitin copolyesters slightly decreases with increase of the acetyl groups content in polymer chain, but it is still high enough to be taken into consideration for eventual production of fibers or scaffolds. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Cellulose/chitosan hybrid nanofibers from electrospinning of their ester derivatives

A facile approach has been established to generate cellulose/chitosan hybrid nanofibers with full range of compositions by electrospinning of their ester derivatives, cellulose acetate (CA) and dibutyryl chitin (DBC), followed by alkaline hydrolysis to cellulose (Cell) and chitosan (CS). DBC was synthesized by acid-catalyzed acylation of chitin (CHI) with butyric anhydride and the newly formed butyl groups on C3 and C6 were confirmed by FT-IR and 1HNMR. DBC had robust solubility in acetone, DMAc, DMF, ethanol, and acetic acid, all except ethanol were also solvents for CA, allowing mixing of these ester derivatives. Fiber formation by electrospinning of either DBC or CA alone and together in these common solvents and their mixtures were studied. The 1/1 acetone/acetic acid was found to be the optimal solvent system to generate fibers from either DBC or CA as well as their mixtures at all CA/DBC ratios, resulting in hybrid fibers with diameters ranging from 30 to 350 nm. DBC and CA were well mixed and showed no phase separate in the hybrid fibers. Alkaline hydrolysis (NaOH) of the equal mass CA/DBC nanofibers regenerated Cell and CHI readily via O-deacylation, then proceeded to further deacetylate CHI to CS via N-deacetylation at higher alkaline concentrations and/or temperatures. Under conditions studied, hydrolysis with 5N NaOH at 100 °C for 3 h was optimal to regenerate cellulose/chitosan hybrid nanofibers.

Radical scavenging activity of 5-methylpyrrolidinone chitosan and dibutyryl chitin

This study compares the behaviour of 5-methylpyrrolidinone chitosan (MPC), a substituted hydrophilic polymer bearing free –OH and –NH 2 groups, and dibutyryl chitin (DBC), a novel lipophilic O- and N-persubstituted chitin, toward free radicals. By means of EPR measurements their capacity of trapping radicals photochemically generated from PTOC esters was assessed. The fate of radicals inside the polysaccharides was checked on the basis of the structure of products obtained after photolysis and characterized by NMR spectroscopy. The experimental data clearly demonstrated that hydrophilic chitosans and persubstituted chitins possess remarkable radical blocking activity even though the nature of the process should be rather different between MPC and DBC. The DBC substrate hosts the radicals in protected and poorly reactive environment, whereas hydrophilic chitosan substrates favor a more rapid annealing of the radicals. MPC takes part in radical reactions with a consequent likely fragmentation of the polymer chain. A photochemical experiment carried out on chitosan confirmed the behaviour observed for MPC.

Effects of Nonwoven Mats of Di-O-butyrylchitin and Related Polymers on the Process of Wound Healing

The aim of the study was to observe the effects of dibutyrylchitin (DBC) on the repair processes and to explain the mechanisms of its action in comparison with other dressing materials made of butyrylchitin (BC), regenerated chitin (RC), and chitosan. The results showed that DBC implanted subcutaneously to the rats increased weight of the granulation tissue. Increased cell number isolated from the wound and cultured on the DBC films was also revealed. The DBC was proved to reduce also the necrotic cells number in the culture. DBC elevates the glycosaminoglycans (GAG) level in the granulation tissue. The total collagen content in the wound was not influenced by all applied dressing materials. However, a low level of the poorly polymerized soluble collagen in the wounds treated with DBC and BC indicated better polymerization of the remaining part of that protein. Both DBC and chitosan increased the weight of granulation tissue. However, chitosan contrary to DBC lowered GAG content and increased water capacity in the wound. The study documents the beneficial influence of DBC on the repair, which could be explained by the modification of the extracellular matrix and cells number. The best effects were observed after application of DBC with [eta] DBC-1 = 1.75 dL/g.

Chitin nanofibrils/chitosan glycolate composites as wound medicaments

Highly crystalline chitin nanofibrils were isolated from crustacean chitin and characterized by X-ray diffraction and FT-infrared spectrometry. A novel formulation including chitin nanofibrils, chitosan glycolate, and chlorhexidine was manufactured in three presentations: spray, gel, and gauze. The latter included non-woven dibutyryl chitin as a biocompatible support. The products were tested in murine wound models, with phytostimuline-medicated wounds as controls, in two series of experiments, one of which included a concomitant laser treatment capable to activate cells. The gauze consistently induced better epithelial differentiation and keratinization, and better reorganization of the basal lamina. The angiogenetic response induced by the gauze was indicative of satisfactory vascular trophism irrespective of the laser treatment. Immunohistochemical observations showed that the actin cytoskeleton of dermal and epidermal cells was well formed. The gauze was used to treat 75 patients hospitalized for a variety of traumatic wounds with good results in all cases. A single dressing could be kept on site for at least four days, and the healing took place within periods of time similar to those reported for traditional dressings under comparable conditions; in no case secondary infections developed. In conclusion, the spray could be used as a first-aid tool on bleeding abrasions; the gel enhanced physiological repair and was recommended for areas with thin epidermal layers, and the gauze was found to be superior to other dressings insofar as no scar remained. A laser treatment could optionally be applied. The biochemical significance of the chitin/chitosan products was found to accompany their commercial viability in terms of time of healing and clinical labor costs.

Changes in structure of dibutyrylchitin fibres in the process of chitin regeneration

Dibutyrylchitin (DBCH) fibres were formed from the solution of the polymer in and anhydrous ethyl alcohol. The fibres obtained were then treated with a 5 % aq. potassium hydroxide solution at temperatures ranging from 20 oC to 90 oC. The process of chitin regeneration was carried out gradually and its rate depended on the temperature. High temperature alkaline treatment causes that the transformation of DBCH fibres into fibres of regenerated chitin becomes more effective. Molecular and supermolecular structural changes of fibres were examined by wide angle X-ray diffraction (WAXS), FT-IR spectroscopy and Raman spectroscopy. The obtained diffractograms were analyzed using Hindeleh and Johnson method and the computer programme "Optifit" in which Rosenbrock method was applied. The analysis of WAXS diffraction patterns showed that the curve obtained for regenerated chitin is similar to the curve obtained for the native krill chitin. The analysis of FT-IR spectra proved that as a result of hydrolysis of DBCH fibres the transformation of DBCH into chitin took place and chemical structure of the obtained chitin was almost the same as the structure of the initial native chitin.

The biocompatibility of dibutyryl chitin in the context of wound dressings

Dibutyryl chitin (DBC) is a modified chitin carrying butyryl groups at 3 and 6 positions; its peculiarity is that it dissolves promptly in common solvents, while being insoluble in aqueous systems. The high biocompatibility of dibutyryl chitin in the form of films and non-wovens has been demonstrated for human, chick and mouse fibroblasts by the Viability/Cytotoxicity assay, In situ Cell Proliferation assay, Neutral Red Retention assay, Lactate Dehydrogenase Release assay, MTS cytotoxicity assay, and scanning electron microscopy. DBC was hardly degradable by lysozyme, amylase, collagenase, pectinase and cellulase over the observation period of 48 days at room temperature, during which no more than 1.33% by weight of the DBC filaments (0.3 mm diameter) was released to the aqueous medium. DBC non-wovens were incorporated into 5-methylpyrrolidinone chitosan solution and submitted to freeze-drying to produce a reinforced wound dressing material. The latter was tested in vivo in full thickness wounds in rats. The insertion of 4×4 mm pieces did not promote any adverse effect on the healing process, as shown histologically. DBC is therefore suitable for contacting intact and wounded human tissues.

Susceptibility of dibutyryl chitin and regenerated chitin fibres to deacylation and depolymerization by lipases

Dibutyryl chitin obtained by esterification with butyric anhydride and regenerated chitin obtained from dibutyryl chitin by saponification, both in the form of wet-spun fibres and non-wovens, were examined by infrared spectrometry and X-ray diffraction spectrometry. Chitin fibres and chitosan fibres were also studied for comparison, and found to maintain the XRD spectral features of the parent chitin and chitosan. On the opposite, DBC fibres and non-woven exhibited depressed crystallinity, the peak at 0.46–0.47 nm, typical of chitin, being hardly detectable, while the one usually at ca. 1.00 nm was present at ca. 1.20 nm. Both DBC fibres and non-woven were highly oriented. When exposed to porcine pancreatic lipase or wheat germ lipase, the DBC fibres gained improved crystallinity with peaks at 1.14–1.18 and 0.41 nm, due to partial regain of chitin structure as a consequence of partial enzymatic removal of butyryl groups, as confirmed by ATR-FTIR. The RC fibres exhibited broad XRD peaks at 0.96 and 0.36 nm; sharper peaks at 0.34, 0.46–0.49 and 0.96 were observed after exposure to lipases, due to removal of a disordered polymer fraction susceptible to the unspecific enzymatic depolymerization. In fact the RC fibres were found to have 8% lower degree of acetylation compared to parent chitin, as a consequence of the alkaline regeneration treatment. In conclusion, these modified chitins are scarcely susceptible to degradation by lipases (besides to lysozyme, as already reported in the literature); therefore their biochemical significance in wound management seems limited. They, however, appear to be the ideal textile materials for providing mechanical support to freeze-dried chitosan sponges having amply documented activity in wound healing, and for the preparation of specialty textiles.

Immiscible Polymer Blends Containing Dibutyrylchitin as Environmentally Friendly Materials

The morphological and physical properties of polymer blends containing dibutyrylchitin (DBCh) are presented in this paper. Two different synthetic polymers, polystyrene (PS) and poly(vinyl acetate) (PVAc) were blended with dibutyrylchitin in common solvent (dimethyl-formamide or methanol) in various weight fractions of DBCh. The films of the blends were prepared by casting method. A polarizing microscope with computer image analyser and differential scanning calorimeter (DSC) were used to evaluate the phase morphology. Microscopic studies indicate heterogeneous structures of both blends. DSC studies show that a characteristic endothermic peak observed for DBCh influenced by water moisturizing effect is shifting toward lower temperatures with an increasing content of the synthetic polymers. The values of glass transition temperatures Tg of both synthetic polymers were found weakly affected by the presence of DBCh.