Enzymatic hydrolysis of chitosan films in water and physiological solution

It was shown that the processes of enzymatic hydrolysis of chitosan in aqueous acetic acid and on the surface of chitosan films in a solution of hyaluronidase in acetic acid are described by uniform kinetic constants. Kinetic parameters of enzymatic hydrolysis of the chitosan film samples in water and in physiological solution (Ringer–Locke’s solution) were determined. It was found that the introduction of medicinal agents and low-molecular-weight electrolytes to a chitosan-based film material reduces the rate of enzymatic hydrolysis of the films, which may indicate a possible increase in their service life when used on the wound surface.     

Growth of human mesenchymal stem cells (MSCs) on films of enzymatically modified chitosan

Mesenchymal stem cells (MSCs) are known to be an attractive cell source for tissue engineering and regenerative medicine. One of the main limiting steps for clinical use or biotechnological purposes is the expansion step. The research of compatible biomaterials for MSCs expansion is recently regarded as an attractive topic. The aim of this study was to create new functional biomaterial for MSCs expansion by evaluating the impact of chitosan derivative films modified by enzymatic approach. First, chitosan particles were enzymatically modified with ferulic acid (FA) or ethyl ferulate (EF) under an eco‐friendly procedure. Then, films of chitosan and its modified derivatives were prepared and evaluated by physicochemical and biological properties. Results showed that the enzymatic grafting of FA or EF onto chitosan significantly increased hydrophobic and antioxidant properties of chitosan films. The MSCs cell viability on chitosan derivative films also increased depending on the film thickness and the quantity of grafted phenols. Furthermore, the cytotoxicity test showed the absence of toxic effect of chitosan derivative films towards MSCs cells. Cell morphology showed a well attached and spread phenotype of MSCs cells on chitosan derivative films. On the other hand, due to the higher phenol content of FA‐chitosan films, their hydrophobic, antioxidant properties and cell adhesion were improved in comparison with those of EF‐chitosan films. Finally, this enzymatic process can be considered as a promising process to favor MSCs cell growth as well as to create useful biomaterials for biomedical applications especially for tissue engineering. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:491–500, 2016     

Enzymatic grafting of hexyloxyphenol onto chitosan to alter surface and rheological properties

An enzymatic method to graft hexyloxyphenol onto the biopolymer chitosan was studied. The method employs tyrosinase to convert the phenol into a reactive o-quinone, which undergoes subsequent nonenzymatic reaction with chitosan. Reactions were conducted under heterogeneous conditions using chitosan films and also under homogeneous conditions using aqueous methanolic mixtures capable of dissolving both hexyloxyphenol and chitosan. Tyrosinase was shown to catalyze the oxidation of hexyloxyphenol in such aqueous methanolic solutions. Chemical evidence for covalent grafting onto chitosan was provided by three independent spectroscopic approaches. Specifically, enzymatic modification resulted in (1) the appearance of broad absorbance in the 350-nm region of the UV/vis spectra for chitosan films; (2) changes in the NH bending and stretching regions of chitosan's IR spectra; and (3) a base-soluble material with (1)H-NMR signals characteristic of both chitosan and the alkyl groups of hexyloxyphenol. Hexyloxyphenol modification resulted in dramatic changes in chitosan's functional properties. On the basis of contact angle measurements, heterogeneous modification of a chitosan film yielded a hydrophobic surface. Homogeneously modified chitosan offered rheological properties characteristic of associating water-soluble polymers.     

Surface structure of chitosan and hybrid chitosan-amylose films-restoration of the antibacterial properties of chitosan in the amylose film

The surface structure of films prepared by casting aqueous solutions of mixtures of water soluble chitosan (WSC) and amylose as well as a fully deacetylated chitosan was studied. Zeta potential measurements indicated that the surface of WSC and fully deacetylated chitosan films is positively charged but very weakly, whereas, a film of amylose blended with WSC exhibited an obvious positive charge. X-ray photoelectron spectra of these films suggest that less amino groups are exposed on the surface of WSC and fully deacetylated chitosan films, whereas, more amino groups are exposed on the surface of a WSC film blended with amylose. A sheet structure in which free amino groups are less exposed on the surface of the film of WSC or fully deacetylated chitosan is proposed. This accounts for the loss of antibacterial activity of chitosan on the WSC film surface. When blended with amylose, the morphology of the film may be disrupted, resulting in strong antibacterial properties.     

Adhesion and growth of HUVEC endothelial cells on films of enzymatically functionalized chitosan with phenolic compounds

Human Umbilical Vein Endothelial Cell (HUVEC) growth on chitosan films and its enzymatically functionalized derivatives films with ferulic acid (FA) and ethyl ferulate (EF) was assessed by evaluating cell adhesion, morphology and cell viability. The results indicated that chitosan derivative films improved protein adsorption properties compared to chitosan films. The HUVEC cell morphology showed well attachment and spread phenotype on chitosan derivative films compared to those growing on chitosan films which did not spread and remained round. Evaluation of cell viability revealed improvement of cell adhesion on chitosan derivative films compared to chitosan film depending on the quantity of oxidized phenols grafted on chitosan. In addition, FA-/EF-chitosan films allowed almost similar cell adhesion. Furthermore, cell adhesion was increased with the film thickness. These results suggested that the oxidized phenols grafting on chitosan is a promising process to enhance cell adhesion, growth and creating useful functional biomaterials.     

Adsorption of chitosan on PET films monitored by quartz crystal microbalance

The adsorption behavior of chitosan on poly(ethylene terephthalate) (PET) model film surface was studied using the quartz crystal microbalance (QCM) technique. QCM with a dissipation unit (QCM-D) represents a very sensitive technique for adsorption studies at the solid/liquid interface in situ, with capability of detecting a submonolayer of adsorbate on the quartz crystal surface. Chitosan as well as PET were chosen for this study due to their promising biocompatible properties and numerous possibilities to be used in biomedical applications. As a first step, PET foils were activated by alkaline hydrolysis in order to increase their hydrophilicity. Model thin films were prepared from PET foils by the spin coating technique. The chemical composition of the obtained model PET films was analyzed using X-ray photoelectron spectroscopy (XPS) and their morphology was characterized by atomic force microscopy (AFM). Furthermore, the adsorption behavior of chitosan on these activated PET films and the influence of adsorption parameters (pH, ionic strength and chitosan solution concentration) were investigated in detail. Additionally, the surface chemistry and morphology of the PET films and the chitosan coated PET films were analyzed with XPS and AFM.     

Enhanced resistance of polyelectrolyte multilayer microcapsules to pepsin erosion and release properties of encapsulated indomethacin

Polyelectrolyte multilayer films were prepared through layer-by-layer (LbL) self-assembly using polysaccharide sodium alginate (ALG) and chitosan (CHI). After incubation in an enzyme pepsin solution, the multilayer film was partially destroyed as detected by the decrease in fluorescent intensity because of the enzymatic degradation of CHI. The enzymatic desorption was also observed from the microcapsule wall made of the ALG/CHI multilayer film directly deposited on indomethacin (IDM) microcrystals through LbL self-assembly. After pepsin erosion, the IDM release from the microcapsules monitored by UV absorbance was obviously accelerated because of desorption. To enhance the stability of the ALG/CHI multilayer film to the enzymatic erosion, some physical and chemical methods were established to increase film thickness or to cross-link the polysaccharides within the film. Increasing the layer number and raising the deposition temperature effectively slowed down the enzymatic desorption and release rate. Especially, increasing deposition temperature was more effective because of producing a more perfect structure in the ALG/CHI multilayer film. Cross-linking the neighboring layers of ALG and CHI with 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide in the ALG/CHI multilayer film significantly reduced the enzymatic desorption and release rate. Therefore, increasing deposition temperature and cross-linking neighboring layers are effective methods to protect the multilayer film fabricated using LbL assembly from the enzymatic erosion and to prolong the release of the encapsulated drug.     

Chitosan film as rhBMP2 carrier: delivery properties for bone tissue application

Tissue engineering approaches need biomaterials with suitable properties to provide an appropriate environment for cell attachment and growth. The performance of these biomaterials can be greatly enhanced through the incorporation of bioactive agents. For this reason, we developed chitosan films with cell-attachment ability, rhBMP-2 carrier capacity, and good in vivo performance, and we employ them as covering for implantable materials. In this work, we have tried to explain how the rh-BMP2 is delivered to the surroundings from the development chitosan films. Protein diffusion from film, film stability versus in vitro dissolution, and biodegradation were evaluated to study rhBMP-2 delivery. Our results show that chitosan film has sufficiently good features to be used as an rhBMP-2 carrier. A low diffusion rate was observed, which was sufficient to quickly induce an in vitro differentiation stimulus, although heavily activated films retain more than 80-85% of the protein on the film. On the other hand, we estimated that chitosan film dissolution due to initial acidification in the wound environment is no more than 15-20%. We also estimated chitosan film response to lysozyme and concluded that degradation via this process proceeded at a slow kinetic rate. In addition, rhBMP-2 in vitro activity after film processing, as well as in vivo film behavior, were studied. We confirm that rhBMP-2 remains active on the film and after release, both in vitro and in vivo. These results support the conclusion that the developed chitosan film allows sustained release of the rhBMP-2 osteoinductive protein and could be used as an activated coat for implant and surgical prosthesis.     

The use of enzymatic preparation for the production of low molecular-weight chitosan from the king crab hepatopancrease

This article describes the optimal conditions for the enzymatic hydrolysis of chitosan and its chemically-modified derivatives using the preparation extracted from the king crab hepatopancrease possessing pronounced hydrolythic activity. The following preparations were used: chitosan with a molecular weight of 700 kDa and an acetylation level of 0.15, carboxymethyl chitosan 200 kDa witih an extent of replacement of 0.23, and N-succinyl chitosan 390 kDa with an extent of replacement of 0.8. Low molecular-weight samples of chitosan and of its modified derivatives were obtained with the yields of 85, 55, and 80%, respectively. The conditions of the hydrolysis were as follows: an enzyme: substrate ratio of 1: 200, 37°C, and 20 h duration of hydrolysis.     

Growth and osteogenic differentiation of adipose-derived and bone marrow-derived stem cells on chitosan and chitooligosaccharide films

Very low molecular weight chitooligosaccharide (COS, 1.4 kDa) and high molecular weight chitosan (1000 kDa) were comparatively studied in terms of physical and biological characteristics. Thin films of COS, chitosan and gelatin were prepared and crosslinked by dehydrothermal treatment at 140 °C for 24 h. COS film presented more hydrophilic property than chitosan film. Behaviors of rat adipose-derived stem cells (ASCs) and bone marrow-derived stem cells (MSCs) were investigated on COS and chitosan films, comparing to those on gelatin film. The results on cell spreading suggested that both ASCs and MSCs preferred to attach on COS film than chitosan film with 6–7 times larger cell areas. Numbers of both stem cells proliferated on COS film were approximately 3-fold higher than those on chitosan film. In addition, COS film enhanced osteogenic differentiating potential of MSCs, as observed from the alkaline phosphatase activity and calcium deposition. Therefore, in this work, COS was shown to be a more favorable material for the growth and osteogenic differentiation of both ASCs and MSCs, compared to high molecular weight chitosan.     

Chitooligosaccharides enzymatic production by Metarhizium anisopliae

The products of chitosan hydrolysis are chitooligosaccharides and are used mainly for medical applications due to their specific biological activities. The objective of this study was to detect and identify the products of enzymatic hydrolysis of chitosan (dimers to hexamers) using a crude extract of chitosanolytic enzymes produced by the fungus Metarhizium anisopliae. These fungus was able to produce, during 48 h cultivation in a medium containing chitosan, chitooligosaccharides ranging from dimers, trimers, tetramers and pentamers at concentrations 0.2, 0.19, 0.06, 0.04 mg/mL, respectively, and the enzymatic activity was 2.5 U/L. Using the crude enzyme extract for chitosan hydrolysis, we detected the presence of dimers to hexamers at hydrolysis times of 10, 20, 30, 40, 50 and 60 min of enzymatic reaction, but the yields were higher at 10 min (54%). The hexamers was obtained only with 30 min of reaction with concentration of 0.004 mg/mL.     

Biodegradable cellulose diacetate-graft-poly(L-lactide)s: enzymatic hydrolysis behavior and surface morphological characterization

Enzymatic hydrolysis of selected copolymers of cellulose diacetate-graft-poly(L-lactide)s (CDA-g-PLLAs) were conducted with proteinase K for film specimens, which were solely quenched from the molten state or, further, annealed at temperatures below or above their glass transition temperatures. The hydrolysis rates depended seriously on the thermal history, as well as on the graft modification. Especially, the heat treatment, followed by physical aging or crystallization of the originally amorphous materials, was a key factor to control subtly their enzymatic degradation behavior. Atomic force microscopy revealed that the enzymatic hydrolysis transformed the surface of the respective films into a more undulated one with a number of fine protuberances, for example, of several hundred nanometers in height and a few micrometers in width. Attenuated total reflection FTIR spectroscopy ensured selective release of lactyl units from the surface region. In visual appearance, some degraded films exhibited even an iridescent color due to an effect of interference of visible light reflected on the surface. These observations suggest a conception of "spatiotemporally controlled degradation", leading to a new method not only for regulation of the overall rate of degradation but also for fine surface abrasion of polymer materials.     

Blends of aliphatic polyesters. VI. Lipase-catalyzed hydrolysis and visualized phase structure of biodegradable blends from poly(epsilon-caprolactone) and poly(L-lactide)

Phase-separated biodegradable polymer blends were prepared from poly(epsilon-caprolactone) (PCL) and poly(L-lactide) (PLLA), and Rhizopus arrhizus lipase-catalyzed hydrolysis and phase structure of the blend films were investigated. Gravimetry revealed that the lipase-catalyzed hydrolysis of PCL in PCL- and PLLA-rich phases is disturbed by the presence of PLLA. Polarimetry confirmed the occurrence of a predominant hydrolysis of PCL and subsequent removal of the hydrolyzed water-soluble PCL oligomers in the blend films. Gravimetry and gel permeation chromatography of the non-blended PLLA film indicated that R. arrhizus lipase has no catalytic effect on the hydrolysis of PLLA. The phase structure of the blend films could be visualized by selective enzymatic removal of one component and subsequent scanning electron microscopic observation.     

Effect of the Degree of Acetylation of Chitosan on Its Enzymatic Hydrolysis with the Preparation Celloviridin G20kh

The degree of acetylation was shown to exert only insignificant effects on the enzymatic hydrolysis of chitosan, while affecting the composition of the resulting hydrolysates and their water solubility. Chitosan with various degrees of acetylation was produced by reacetylation of the initial chitosan (the solvents, methanol and 2% acetic acid, were present in a ratio of 54 : 51 v/v; the amount of acetic anhydride was in the range 0.1–2.0 mmol per gram chitosan). Hydrolysis by the enzymatic preparation Celloviridin G20kh was performed at an enzyme-to-substrate ratio of 1 : 400 in sodium–acetate buffer, pH 5.2 (55°C) for 1 h.     

Chitosan-vancomysin composite biomaterial as a laser activated surgical adhesive with regional antimicrobial activity

We have used laser irradiation to enhance the natural adhesiveness of chitosan to form a thin film surgical adhesive. Prevention of infection at surgical sites often utilizes systemic provision of antibiotics with reduced local efficacy and potential side effects. In the work reported here, we investigate the bactericidal properties of laser-irradiated chitosan films and their impregnation with the antibiotic vancomycin. Despite strong efficacy in solution, chitosan films showed no antimicrobial activity against representatives of common pathogens Escherichia coli , Staphylococcus aureus , and S. epidermidis . In contrast, a composite of chitosan adhesive and the antibiotic vancomycin showed therapeutically significant release profiles greater that the Minimum Bactericidal Concentrations (MBCs) for the Staphylococci over a 28 day period. These composite films had greater crystallinity, up to 28 ± 3 compared to 8.9 ± 2%, for its unblended counterpart. Despite a significant increase in material strength from 31.4 ± 4 to 77.5 ± 5 MPa, flexibility was still maintained with an elongation to break around 5 ± 2% and fold endurance of approximately 30 ± 3-folds. Laser irradiation had no apparent effect on the release or activity of the antibiotic which survived transient temperatures at the film-tissue interface during infrared irradiation of around 54 °C. Furthermore, significant adhesive strength was still apparent, 15.6 ± 2 KPa. Thus, we have developed a laser-activated bioadhesive with the potential to close wounds while facilitating the prevention of microbial infection through local release of antibiotic targeted to the site of potential infection.     

Effect of addition of water-soluble chitin on amylose film

Amylose films blended with chitosan, which were free from additives such as acid, salt, and plasticizer, were prepared by casting mixtures of an aqueous solution of an enzymatically synthesized amylose and that of water-soluble chitin (44.1% deacetylated). The presence of a small amount of chitin (less than 10%) increased significantly the permeability of gases (N2, O2, CO2, C2H4) and improved the mechanical parameters of amylose film; particularly, the elastic modulus and elongation of the blend films were larger than those of amylose or chitin films. No antibacterial activity was observed with either amylose or water-soluble chitin films. But amylose films having a small amount of chitin showed strong antibacterial action, suggesting a morphological change in water-soluble chitin on the film surface by blending with amylose molecule. These facts suggested the presence of a molecular complex of amylose and chitosan.     

Hydrolysis of chitosan sulfate by an enzyme complex from Streptomyces kurssanovii

The possibility of enzymatic hydrolysis of chitosan was shown. The optimum conditions for the process are: sodium acetate buffer pH 6.0, 37 degrees C, 24 h, and the chitosan sulfate-protein volume ratio of 500:1 in the enzyme preparation. During hydrolysis, the intrinsic viscosity of chitosan sulfate solution decreased by a factor of 2.7.     

Depolymerization of chitosan by chinolytic complex from Bacillus sp. 739

Low-molecular-weight (3-6 kDa) water-soluble chitosan was obtained by enzymatic depolymerization. Hydrolysis of crab chitosan was induced by O-glycoside hydrolase (EC 3.2.1), an extracellular chitinolytic complex from Bacillus sp. 739. The optimum conditions for hydrolysis were found (sodium-acetate buffer, pH 5.2; 55 degrees C; an enzyme/substrate ratio 4 U/g chitosan; 1 h).     

Condensed state structure and biocompatibility of the konjac glucomannan/chitosan blend films

Specialised blend films have been prepared by blending 1% w/v konjac glucomannan aqueous with 1% w/v chitosan solution in acetate solution and drying at room temperature for 24 h. The condensed state structure and miscibility of the blend films were studied by Fourier transform infrared spectroscopy, scanning electron microscopy, differential scanning calorimetry, and wide-angle X-ray diffraction. The results indicated that the blend film obtained from an 80/20 mixing ratio of konjac glucomannan and chitosan derivate showed the highest miscibility and blend homogeneity, and that strong intermolecular hydrogen bonds took place between the amino groups of chitosan and the hydroxyl groups of konjac glucomannan; thus the tensile strength also achieved its maximum in this ratio. The cell morphologies on the pure and blend films were examined by light microscopy and cell viability was studied by using MTT assay. The results showed that the particular blend film was more suitable for the cell culture than the pure konjac glucomannan film, and that the cells cultured on this blend film had greater spreading coefficients than that of the pure konjac glucomannan film. As a result of the good mechanical properties, miscibility and biocompatibility, the blend film is a promising biomaterial matrix.     

Degradability of polysaccharides multilayer films in the oral environment: an in vitro and in vivo study

Biomedical devices and modified biomaterial surfaces constitute an expanding research domain in the dental field. However, such oral applications have to face a very particular environment containing specific physiological conditions and specific enzymes. To evaluate their suitability in the development of novel oral applications, the degradability of polyelectrolyte multilayer films made of the natural polysaccharides chitosan and hyaluronan (CHI/HA) was investigated in vitro and in vivo in a rat mouth model. The films were either native or cross-linked using a water-soluble carbodiimide (EDC) in combination with N-hydroxysulfosuccinimide. The in vitro degradation of the films by different enzymes present in the oral environment, such as lysozyme and amylase, was followed by quartz crystal microbalance measurements and confocal laser scanning microscopy observations after being film labeled with CHI(FITC). Whereas native films were subjected to degradation by all the enzymes, cross-linked films were more resistant to enzymatic degradation. Films were also put in contact with whole saliva, which induced a slow degradation of the native films over an 18 h period. The in vivo degradation of the films deposited on polymer disks and sutured in the rat mouth was followed over a 3 day period. Whereas film degradation is fast for native films, it is much slower for the cross-linked ones. More than 60% of these films remained on the disks after 3 days in the mouth. Taken together, these results suggest that the multilayer films made of natural polysaccharides are of high potential interest for oral applications, especially as drug release systems, offering various degradation rates and consequent release characteristics.