Physicochemical and structural characteristics of chitosan nanopowders prepared by ultrafine milling

Two different molecular weights of chitosan were pulverized to nanopowders by ultrafine milling. The nanopowders were characterized by viscometry small angle X-ray scattering (SAXS), transmission electron microscopy (TEM), X-ray diffraction (XRD), thermogravimetric analysis (TGA), FT-IR spectroscopy and UV-vis spectroscopy. Our results showed that ultrafine milling effectively reduced the particle size of chitosan to a nanoscale. The viscosity average molecular weight (Mv) of chitosan was decreased by the milling treatment. The crystalline structure of chitosan was destroyed by the milling since the nanopowder exhibited an amorphous XRD pattern. In addition, thermal stability of the low molecular weight chitosan was decreased after the milling treatment. FT-IR and UV-vis spectra showed that the milling process did not cause significant changes in the chemical structure of chitosan.     

Immobilization of a protease on modified chitosan beads for the depolymerization of chitosan

Neutral protease was immobilized on chitosan (CS), carboxymethyl chitosan (CMCS), and N-succinyl chitosan (NSCS) hydrogel beads. And the biocatalysts obtained were used to prepare low molecular weight chitosan (LMWC) and chitooligomers. Weight-average molecular weight of LMWC produced by neutral protease immobilized on CS, CMCS and NSCS hydrogel beads were 3.4 kDa, 3.2 kDa and 1.9 kDa, respectively. The effects of immobilization support and substrate on enzymatic reaction were analyzed by measuring classical Michaelis-Menten kinetic parameters. The FT-IR, XRD and potentiometric determination results indicated decrease of molecular weight led to transformation of crystal structure, but the degree of N-deacetylation and chemical structures of residues were not changed compared to initial chitosan. The degree of polymerization of chitooligomers was mainly from 2 to 7. We observed a strong dependence of the immobilized enzyme properties on the chemical nature of the supports, which leads to different microenvironment of neutral protease and changes the hydrolyzing process.     

Lipase-coupling esterification of starch with octenyl succinic anhydride

Enzymatic modification of starch was conducted by lipase-coupling esterification with octenyl succinic anhydride (OSA). Parameters affecting the esterification were systematically studied. Products were characterized by Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction, differential scanning calorimetry (DSC) and viscosity analysis (VA). Optimum condition for lipase-coupling OSA starch preparation was as follows: starch pretreatment at 65 °C for 15 min, starch concentration 35%, amount of lipase and OSA, 0.6% and 3%, reaction pH, temperature and time, 8.0, 40 °C and 30 min respectively, which resulted in 0.0195 of the degree of substitution and 84.05 ± 2.07% of the reaction efficiency. FT-IR spectroscopy confirmed the formation of OSA starch. SEM and X-ray diffraction showed apparent surface change, but no crystalline change. DSC and VA results indicated the synthesized OSA starch gelatinized rapidly with high viscosity. Attractively, reaction time drastically reduced to 30 min, showing vast potential for scale production of OSA starch.     

Anti-HIV-1 activity of low molecular weight sulfated chitooligosaccharides

Chitooligosaccharides are nontoxic and water-soluble compounds obtained by enzymatic degradation of chitosan, which is derived from chitin by a deacetylation process. Chitooligosaccharides possess broad range of activities such as antitumour, antifungal, antibacterial activities. Sulfated chitooligosaccharides (SCOSs) with different molecular weights were synthesized by a random sulfation reaction. In the present study, anti-HIV-1 properties of SCOSs and the impact of molecular weight on their inhibitory activity were investigated. SCOS III (MW 3-5 kDa) was found to be the most effective compound to inhibit HIV-1 replication. At nontoxic concentrations, SCOS III exhibited remarkable inhibitory activities on HIV-1-induced syncytia formation (EC₅₀ 2.19 μg/ml), lytic effect (EC₅₀ 1.43 μg/ml), and p24 antigen production (EC₅₀ 4.33 μg/ml and 7.76 μg/ml for HIV-1_RF and HIV-1_Ba-L, respectively). In contrast, unsulfated chitooligosaccharides showed no activity against HIV-1. Furthermore, it was found that SCOS III blocked viral entry and virus-cell fusion probably via disrupting the binding of HIV-1 gp120 to CD4 cell surface receptor. These results suggest that sulfated chitooligosaccharides represent novel candidates for the development of anti-HIV-1 agent.     

Antimicrobial textile treated with chitosan from Aspergillus niger mycelial waste

The waste biomass of Aspergillus niger, following citric acid production, was used as a source for fungal chitosan extraction. The produced chitosan was characterized with deacetylation degree of 89.6%, a molecular weight of 25,000 dalton, 97% solubility in 1% acetic acid solution and comparable FT-IR spectra to standard shrimp chitosan. Fungal chitosan was applied as a cotton fabric finishing agent using pad-dry-cure method. The topographical structure of chitosan-treated fabrics (CTF) was much improved compared with control fabrics. CTF, after durability tests, exhibited a powerful antimicrobial activity against both E. coli and Candida albicans, the captured micrographs for E. coli cells contacted with CTF showed a complete lysis of cell walls with the prolonging contact time. The produced antimicrobial CTF could be proposed as a suitable material for many medical and hygienic applications.     

Mucoadhesive chitosan/gelatin films for buccal delivery of propranolol hydrochloride

The aim of this work was to develop and characterize chitosan/gelatin films as innovative mucoadhesive system for buccal delivery of propranolol hydrochloride. FT-IR and TGA analysis confirmed the interaction between chitosan and gelatin. The presence of higher chitosan amounts in chitosan/gelatin films allowed the lowest percent water-uptake ability (235.1 ± 5.3%) and the highest in vivo residence time in the buccal cavity (240 ± 13 min). Moreover, the presence of mannitol in the formulation allowed 80% drug permeation through porcine buccal mucosa in 5 h. This behaviour suggests that the application of four and two films containing 5 mg of propranolol hydrochloride could be suitable for achieving the proposed daily dose for hypertension and atrial fibrillation treatment, respectively. Another interesting aspect of chitosan/gelatin films was their compatibility with buccal microflora in the absence of drug and their ability to determine growth inhibition for pathogen bacteria, but not for probiotic species, when loaded with drug.     

Chitosan-LiOH-urea aqueous solution—a novel water-based system for chitosan processing

A solution of partially N-deacetylated chitosan in aqueous lithium hydroxide (LiOH)/urea was prepared successfully through a freeze-thawing process and the dissolution behavior was studied. The results indicated that chitosan can directly dissolve in LiOH/urea aqueous solution. LiOH mainly contributed to the breakage of intramolecular and intermolecular hydrogen bonds in chitosan. Urea, LiOH, and chitosan formed inclusion compound (IC) with urea as the IC host, and the LiOH-chitosan complex as the guest. Aqueous 4.8 wt % LiOH/8.0 wt % urea was verified to be the optimal solvent for chitosan. The results of rheology and viscosity characterizations revealed that chitosan/4.8 wt % LiOH/8.0 wt % urea aqueous solution was pseudoplastic fluid, and was more stable than the solution of chitosan in acetic acid at ambient temperature.     

Rheological properties of chitosan-tripolyphosphate complexes: From suspensions to microgels

Complex fluids formed by crosslinking of chitosan (CS, 330 kDa) with sodium tripolyphosphate (TPP) have been studied by dynamic light scattering (DLS), atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR), and rheology. The effects of chitosan/TPP ratios, initial chitosan or TPP concentrations, and ultrasonication time on the chitosan-TPP complex formation have been investigated. It was found that the optimum condition for CS-TPP nanoparticle formation occurred at CS/TPP mass ratio of 3.75 and with 9 min sonication treatment (energy output 3.75 W/mL). At the same initial chitosan concentration, small particle sizes (i.e., particle size < 300 nm) resulted in the formation of CS-TPP nanoparticle suspensions, which showed a lower viscosity than pure chitosan solutions, and their viscosities increased as the CS-TPP nanoparticles sizes increased. Centrifugation of CS-TPP particles of larger particle sizes (i.e., 360-870 nm) at 11,000 × g caused the formation of CS-TPP microgels. Dynamic rheological studies indicated that both storage modulus (G′) and loss modulus (G″) increased with particle sizes. During centrifugation processing, strong centrifugal force surmounted the electrostatic repulsion between CS-TPP particles and caused particles to stick with each other to form CS-TPP microgels. The water contents of microgels negligibly depended on particle size, suggesting that the free volumes of microgels were not affected by particle size, therefore supporting our pseudo-hard sphere assumption for CS-TPP nanoparticles.     

Cross-linked succinyl chitosan as an adsorbent for the removal of Methylene Blue from aqueous solution

Removal of a basic dye (Methylene Blue) from aqueous solution was investigated using a cross-linked succinyl-chitosan (SCCS) as sorbent. The chemical structures of chitosan and its derivatives were testified by FT-IR. X-ray diffraction, DTG analysis and swelling measurements were conducted to clarify the characteristics of the chemically modified chitosan. The effect of process parameters, such as pH of the initial solution, and concentrations of dyes on the extent of Methylene Blue (MB) adsorption was investigated. The Langmuir isotherm model was used to fit the equilibrium experimental data, giving a maximum sorption capacity of 289.02 mg/g at 298 K. Kinetic studies showed that the kinetic data were well described by the pseudo-second-order kinetic model. Thermodynamic parameters such as enthalpy change (ΔH°), free energy change (ΔG°) and entropy change (ΔS°) were determined to be −25.32 kJ mol⁻¹, −6.76 kJ mol⁻¹ and −62.36 J mol⁻¹ K⁻¹, respectively, which leads to a conclusion that the adsorption process is spontaneous and exothermic.     

Effect of ultrasonic treatment on the biochemphysical properties of chitosan

Four chitosans with different molecular weights and degrees of deacetylation degree and 28 chitosans derived from these initial chitosans by ultrasonic degradation have been characterized by gel permeation chromatography (GPC), FT-IR spectroscopy, X-ray diffraction and titrimetric analyses. Antimicrobial activities were investigated against E. coli and S. aureus using an inhibitory rate technique. The results showed that ultrasonic treatment decreased the molecular weight of chitosan, and that chitosan with higher molecular weight and higher DD was more easily degraded. The polydispersity decreased with ultrasonic treatment time, which was in linear relationship with the decrease of molecular weight. Ultrasonic degradation changed the DD of initial chitosan with a lower DD (<90%), but not the DD of the initials chitosan with a higher DD (>90%). The increased crystallinity of ultrasonically treated chitosan indicated that ultrasonic treatment changed the physical structure of chitosan, mainly due to the decrease of molecular weight. Ultrasonic treatment enhanced the antimicrobial activity of chitosan, mainly due to the decrease of molecular weight.     

Inter-connecting pores of chitosan scaffold with basic fibroblast growth factor modulate biological activity on human mesenchymal stem cells

In this study, we developed bio-active molecules immobilized chitosan scaffolds with controlled pore architectures for enhanced viability of human mesenchymal stem cells (hMSCs). The decreasing in molecular weight of chitosan by ultrasonication of chitosan solution was effective in the formation of porous chitosan scaffolds, resulting in an increase of inter-connecting micropores (∼10 μm) between macropores. Using a layer-by-layer method, we then prepared heparin-coated chitosan scaffolds as depots for basic fibroblast growth factors (bFGF). Enzyme-linked immunosorbent assays confirmed that heparin-coated chitosan scaffolds could bind higher amount of bFGF (24.21 ng/mg) compared to 2.53 ng/mg of non-coated scaffold. Moreover, we were able to manipulate the release profiles of bFGF from the scaffolds for 7 days. In vitro studies showed that chitosan scaffolds induced the improved viability and proliferation of hMSCs through their synergetic effects of the inter-connecting micropores and the sustained released of bFGF. Our results suggest that bFGF immobilized chitosan scaffolds with controlled inter-connecting pores, in combination with other heparin-binding growth factors, have potential implants for controlling biological functions in regenerative medicine.     

Biocomposite films prepared from ionic liquid solutions of chitosan and cellulose

Blends of chitosan and cellulose were successfully produced using 1-butyl-3-methylimidazolium acetate (BMIMAc) as solvent media. Films were prepared from the blends by manually spreading the solution on a flat surface and precipitating the polymers in a mixture of methanol and water. To prevent the shrinkage of films, most of the absorbed water was removed by freeze drying under vacuum. Films prepared from the polymeric solutions were investigated by means of FT-IR, TGA, X-ray diffraction and SEM measurements. The shifting of the bands corresponding to -NH and CO groups of chitosan (FT-IR), the absence of the diffraction peaks at 2θ = 10.7 and 14.9° (XRD), the increased E_a for thermal decomposition for all the polymeric blends (MTGA), and the presence of an apparent homogeneous structure with no phase separation of the two polymers (SEM) provide evidence for the miscibility between chitosan and cellulose in the solid state.     

Preparation and characterization of molecular weight fractions of glycosaminoglycan from sea cucumber Thelenata ananas using free radical depolymerization

A glycosaminoglycan from sea cucumber Thelenata anana (THG) was isolated as a polymer of molecular weight of around 70 kDa. Its low molecular weight derivatives were first prepared by free radical depolymerization with hydrogen peroxide in the presence of copper(II) ion. The parameters of the process were investigated by a high-performance gel permeation chromatography. Analyses of chemical composition and molecular weight distribution indicated that the fragmentation of the main-chain of THG occurred randomly, obeyed pseudo first-order kinetics, and produced species with rather narrow and unimodal distribution of molar mass. The characterization of different molecular weight fractions was investigated by using viscometry and atomic force microscopy (AFM). Analysis of molecular weight and intrinsic viscosity in terms of the known theories for unperturbed wormlike cylinder yielded 1201 ± 110 nm⁻¹, 15.3 ± 1.5 nm, and 1.5 ± 0.3 nm for molar mass per unit contour length M_L, persistence length q, and diameter d, respectively. The M_L and d values were approximately consistent with those observed by AFM. The present data suggest that THG may dissolve in 0.1 M aqueous NaCl as single-stranded helical chains.     

Interaction of chitosans and their N-acylated derivatives with lipopolysaccharide of gram-negative bacteria

The interactions of lipopolysaccharide (LPS) with the natural polycation chitosan and its derivatives--high molecular weight chitosans (80 kD) with different degree of acetylation, low molecular weight chitosan (15 kD), acylated oligochitosan (5.5 kD) and chitooligosaccharides (biose, triose, and tetraose)--were studied using ligand-enzyme solid-phase assay. The LPS-binding activity of chitosans (80 kD) decreased with increase in acetylation degree. Affinity of LPS interaction with chitosans increased after introduction of a fatty acid residue at the reducing end of chitosan. Activity of N-monoacylated chitooligosaccharides decreased in the order: oligochitosan --> tetra- > tri- --> disaccharides. The three-dimensional structures of complexes of R-LPS and chitosans with different degree of acetylation, chitooligosaccharides, and their N-monoacylated derivatives were generated by molecular modeling. The number of bonds stabilizing the complexes and the energy of LPS binding with chitosans decreased with increase in acetate group content in chitosans and resulted in changing of binding sites. It was shown that binding sites of chitooligosaccharides on R-LPS overlapped and chitooligosaccharide binding energies increased with increase in number of monosaccharide residues in chitosan molecules. The input of the hydrophobic fragment in complex formation energy is most prominent for complexes in water phase and is due to the hydrophobic interaction of chitooligosaccharide acyl fragment with fatty acid residues of LPS.     

Ozone gas affects physical and chemical properties of wheat (Triticum aestivum L.) starch

In this experiment, bread wheat flour and isolated wheat starch were treated with ozone gas (1,500 mg/kg at 2.5 L/min) for 45 min and 30 min, respectively. Starch was isolated from treated flour. Ozone treated starch and starch isolated from ozone treated flour had similar chemical and physical properties. Chemical analysis of starch isolates indicated depolymerization of high molecular weight amylopectins; with a subsequent increase in low molecular weight starch polymers as a result of starch hydrolysis. Ozone treatment resulted in elevated levels of carboxylic groups and decreased total carbohydrate content in amylopectin fractions. ¹H NMR results indicated formation of a keto group [(1→4)-3 keto] at the H-2 terminal (proton at C-2 position) and β-glucuronic acid at the H-1 terminal (proton at C-1 position). DSC transition temperatures and change in enthalpy were not affected by ozone treatment. Increased swelling power and RVA breakdown were observed in starch from ozone treated samples.     

Study on the heterogeneous degradation of chitosan with H2O2 catalyzed by a new supermolecular assembly crystal: [C6H8N2]6H3[PW12O40]·2H2O

A new supermolecular assembly crystal, [C₆H₈N₂]₆H₃[PW₁₂O₄₀]·2H₂O (DMB-PWA), was synthesized with phosphotungstic acid (PWA) and 1,2-diaminobenzene (DMB) under hydrothermal conditions and was characterized by Fourier-transform infrared spectra (FTIR) and single-crystal X-ray diffraction analysis. DMB-PWA could effectively catalyze oxidative degradation of chitosan with H₂O₂ in the heterogeneous phase. The optimum degradation conditions were determined by orthogonal tests as follows: amount of chitosan 1.00 g, 30% (wt %); H₂O₂, 3.0 mL; dosage of catalyst, 0.06 g; reaction temperature, 85 °C; and reaction time, 30 min. The water-soluble chitosan with a viscosity-average molecular weight (M_v) of 4900 was obtained under the optimum degradation conditions and was characterized by FTIR, ultraviolet-visible diffuse reflection spectra (UV-vis DRS), and X-ray powder diffraction analysis.     

The cytoprotection of chitosan based hydrogels in xenogeneic islet transplantation: An in vivo study in streptozotocin-induced diabetic mouse

Immune rejection and scarcity of donor tissues are the restrictions of islets transplantation. In this study, the cytoprotection of chitosan hydrogels in xenogeneic islet transplantation was demonstrated. Wistar rat islets encapsulated in chitosan hydrogels were performed glucose challenge test and live/dead cell staining in vitro. Islets/chitosan hydrogels were transplanted into the renal subcapsular space of diabetic C57BL/6 mice. Non-fasting blood glucose level (NFBG), body weight, intraperitoneal glucose tolerance test (IPGTT), and glucose disappearance rate were determined perioperatively. The serum insulin level was analyzed, and the kidney transplanted with islets/chitosan hydrogels were retrieved for histological examination after sacrifice. The present results showed that islets encapsulated in chitosan hydrogels secreted insulin in response to the glucose stimulation as naked islets with higher cell survival. The NFBG of diabetic mice transplanted with islets/chitosan hydrogels decreased from 487 ± 46 to 148 ± 32 at one day postoperation and maintained in the range of 201 ± 36 mg/dl for four weeks with an increase in body weight. IPGTT showed the glucose disappearance rate of mice transplanted with islets/chitosan hydrogels was significant faster than that of mice transplanted with naked islets; the serum insulin level increased from 0.29 ± 0.06 to 1.69 ± 0.65 μg/dl postoperatively. Histological examination revealed that the islets successfully engrafted at renal subcapsular space with positive insulin staining. The immunostain was negative for neither the T-cell lineages nor the monocyte/macrophages. This study indicates that the chitosan hydrogels deliver and protect encapsulated islets successfully in xenotransplantation.     

Effect of reaction temperature and reaction time on the preparation of low-molecular-weight chitosan using phosphoric acid

Low-molecular-weight chitosan were prepared using 85% phosphoric acid at different reaction temperatures and reaction time. At room temperature, the viscosity average-molecular weights (M_v) of chitosan decreased to 7.1×10⁴ from 21.4×10⁴ after 35 days treatment. The degradation rate decreased with increasing hydrolysis time. The yields of chitosan also continuously decreased from 68.4 to 40.2% after 35 days. At 40, 60 and 80 °C, the molecular weight decreased to 3.70×10⁴, 3.50×10⁴ and 2.00×10⁴ on 8 h hydrolysis, respectively. The yields of chitosan remain at a high level compared with that at room temperature and were 86.5, 71.4 and 61.3% at 40, 60 and 80 °C treatment, respectively. The different reaction time gave chitosan with different molecular weights. At 60 °C, the molecular weight of products decreased to 7.40×10⁴ from 21.4×10⁴ within 4 h, then decreased slowly to 1.90×10⁴ in 15 h. It was also found that the water-solubility of chitosan increased as the molecular weight decreased. Results show the changes in yields and molecular weight of chitooligomers were strongly dependent on the reaction temperature and reaction time.     

Effect of carboxymethylation conditions on the water-binding capacity of chitosan-based superabsorbents

A superabsorbent polymer (SAP) from chitosan was provided via carboxymethylation of chitosan, followed by cross-linking with glutaraldehyde and freeze-drying. This work was focused on an investigation of the effects of monochloroacetic acid (MCAA), sodium hydroxide, and reaction time on preparation of carboxymethyl chitosan (CMCS). The CMCS products were characterized using FTIR spectroscopy, and their degrees of substitution (DS) were measured using conductimetry and FTIR analysis. The highest DS value was obtained when the carboxymethylation reaction was carried out using 1.75 g MCAA and 1.75 g NaOH per g of chitosan in 4 h. The water solubilities of the CMCS products at various pHs were also evaluated, and the results indicated a significant impact of the reaction parameters on the solubility of CMCS. The CMCSs with the highest DS value resulted in SAPs having the highest water-binding capacity (WBC). The WBC of the best SAP measured after 10 min exposure in distilled water, 0.9% NaCl solution, synthetic urine, and artificial blood was 104, 33, 30, and 57 g/g, respectively. The WBC of this SAP at pH 2-9 passed a maximum at pH 6.     

Antifungal property of quaternized chitosan and its derivatives

Five water-soluble chitosan derivatives were carried out by quaternizing either iodomethane or N-(3-chloro-2-hydroxypropyl) trimethylammonium chloride (Quat188) as a quaternizing agent under basic condition. The degree of quaternization (DQ) ranged between 28 ± 2% and 90 ± 2%. The antifungal activity was evaluated by using disc diffusion method, minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) methods against Trichophyton rubrum (T. rubrum), Trichophyton mentagrophyte (T. mentagrophyte), and Microsporum gypseum (M. gypseum) at pH 7.2. All quaternized chitosans and its derivatives showed more effective against T. rubrum than M. gypseum and T. mentagrophyte. The MIC and MFC values were found to range between 125-1000 μg/mL and 500-4000 μg/mL, respectively against all fungi. Our results indicated that the quaternized N-(4-N,N-dimethylaminocinnamyl) chitosan chloride showed highest antifungal activity against T. rubrum and M. gypseum compared to other quaternized chitosan derivatives. The antifungal activity tended to increase with an increase in molecular weight, degree of quaternization and hydrophobic moiety against T. rubrum. However, the antifungal activity was depended on type of fungal as well as chemical structure of the quaternized chitosan derivatives.