Preparation and pharmacodynamics of low-molecular-weight chitosan nanoparticles containing insulin

Low-molecular-weight chitosan (LMWC) was obtained by enzymatic degradation and ultrafiltration separation. LMWC nanoparticles with LMWC having 20 kDa weight average molecular weight (M_w) were then prepared by solvent evaporation method. The resultant nanoparticles were spherical with a narrow particle size distribution. LMWC nanoparticles loaded with insulin as a model drug were prepared. The average entrapment efficiency of insulin could reach up to 95.54%. The in vitro drug release profiles from the nanoparticles showed an initial burst of release in the first 2 h, followed by zero order release kinetics. In vivo pharmacodynamics of chitosan nanoparticles containing insulin showed that the nanoparticles showed some hypoglycemic activity. Compared with an insulin solution, a relative bioavailability of 0.737 was observed for four times the dosage of insulin in the chitosan nanoparticles after pulmonary administration.     

Low molecular weight chitosans—Preparation with the aid of pronase,characterization and their bactericidal activity towards Bacillus cereus and Escherichia coli

The homogeneous low molecular weight chitosans (LMWC) of molecular weight 9.5–8.5 kDa, obtained by pronase catalyzed non-specific depolymerization (at pH 3.5, 37 °C) of chitosan showed lyses of Bacillus cereus and Escherichia coli more efficiently (100%) than native chitosan (< 50%). IR and ¹H-NMR data showed decrease in the degree of acetylation (14–19%) in LMWC compared to native chitosan (∼ 26%). Minimum inhibitory concentration of LMWC towards 10⁶ CFU ml^− 1 of B. cereus was 0.01% (w/v) compared to 0.03% for 10⁴ CFU ml^− 1 of E. coli. SEM revealed pore formation as well as permeabilization of the bacterial cells, as also evidenced by increased carbohydrate and protein contents as well as the cytoplasmic enzymes in the cell-free supernatants. N-terminal sequence analyses of the released proteins revealed them to be cytoplasmic/membrane proteins. Upon GLC, the supernatant showed characteristic fatty acid profiles in E. coli, thus subscribing to detachment of lipopolysaccharides into the medium, whereas that of B. cereus indicated release of surface lipids. The mechanism for the observed bactericidal activity of LMWC towards both Gram-positive and Gram-negative bacteria has been discussed.     

The antimicrobial action of low-molar-mass chitosan,chitosan derivatives and chitooligosaccharides on bifidobacteria

The crude fractions of chitooligosaccharides (COS) and low-molar-mass chitosans (LMWC) were prepared by enzyme hydrolysis of chitosan (CS). Specific growth rate of B. adolescentis, B. bifidum, B. breve, B. catenulatum, B. infantis and B. longum ssp. longum was determined in the presence of 0.025 and 0.5 % COS (<5 kDa), LMWC (5–10 kDa), and 0.025, 0.1 and 0.5 % of CS, chitosan succinate and chitosan glutamate in vitro. Minimum inhibitory concentrations (MIC; assayed by colony counting on TPY agar plates) of COS-LMWC and CS ranged from 0.025 % to 0.75 % of CS-LMWC. The growth of all bifidobacterial strains in the presence of chitosan, its derivatives and LMWC decreased at a concentration of 0.025 %; the bacterial growth was completely inhibited at a concentration of 0.5 %. COS did not show any inhibitory effect, an increased growth rate was even observed in the case of B. bifidum, B. catenulatum and B. infantis.     

The antimicrobial action of chitosan,low molar mass chitosan,and chitooligosaccharides on human colonic bacteria

Antibacterial effect of chitooligosaccharides (COS) and low molar mass chitosans (LMWC) is considered as one of the most important characteristics of chitosan (CS) hydrolysates. Here, we show the in vitro effect of different COS, LMWC, and CS on representative anaerobic bacteria isolated from human colon as a possibility of targeting modification of colonic microflora composition by supplementation of dietary CS products by humans. Specific growth rate of seven selected nonpathogenic anaerobic bacterial strains (Clostridium paraputrificum, Clostridium beijerinckii, Roseburia intestinalis, Bacteroides vulgatus, Bacteriodes thetaiotaomicron, Faecalibacterium prausnitzii and Blautia coccoides) was determined in the presence of 0.25 and 0.5% COS (2, 3, and 6 kDa), 0.025 and 0.05% of LMWC (10 and 16 kDa), and 0.025 and 0.1% of CS in vitro. The growth rate decreased in all strains in the presence of COS and LMWC in higher concentrations in comparison to control incubations. A relatively higher resistance to CS hydrolyzates was detected in R. intestinalis and F. prausnitzii, and more susceptible were bacteria belonging to Bacteoides sp. and Clostridium sp. The antimicrobial activity, minimum inhibitory concentrations (MIC), and minimal bactericidal concentrations (MBC) were determined. The antimicrobial activity increased with the degree of polymerization (DP). MIC ranged from 0.25 to 4.5% in dependence on bacterial strain and DP of CS/LMWC. MBC also decreased with DP. The most effective antimicrobial action was detected in LMWC with 16 kDa and CS. Weak antimicrobial activity was found in COS with small molecules (2 and 3 kDa).     

Non-specific depolymerization of chitosan by pronase and characterization of the resultant products.

Pronase (type XXV serine protease from Streptomyces griseus) efficiently depolymerizes chitosan, a linear beta-->1,4-linked polysaccharide of 2-amino-deoxyglucose and 2-amino-2-N-acetylamino-D-glucose, to low-molecular weight chitosans (LMWC), chito-oligomers (degree of polymerization, 2-6) and monomer. The maximum depolymerization occurred at pH 3.5 and 37 degrees C, and the reaction obeyed Michaelis-Menten kinetics with a Km of 5.21 mg.mL(-1) and Vmax of 138.55 nmoles.min(-1).mg(-1). The molecular mass of the major product, LMWC, varied between 9.0 +/- 0.5 kDa depending on the reaction time. Scanning electron microscopy of LMWC showed an approximately eightfold decrease in particle size and characterization by infrared spectroscopy, circular dichroism, X-ray diffractometry and 13C-NMR revealed them to possess a lower degree of acetylation, hydration and crystallinity compared to chitosan. Chitosanolysis by pronase is an alternative and inexpensive method to produce a variety of chitosan degradation products that have wide and varied biofunctionalities.     

In vitro evaluation of the biomedical properties of chitosan and quaternized chitosan for dental applications

The aim of this study was to evaluate the potential dental applications of chitosan (CS) and N-[1-hydroxy-3-(trimethylammonium)propyl]chitosan chloride (HTCC). HTCC was prepared by reacting CS with glycidyltrimethylammonium chloride (GTMAC). CS and HTCC were characterized by infrared (FITR) and ¹H NMR spectroscopy. The antibacterial activity of CS and HTCC against oral pathogens, their proliferation activity and effects on the ultrastructure of human periodontal ligament cells (HPDLCs) were investigated. The results indicated that four oral strains were susceptible to CS and HTCC with minimum inhibitory concentrations (MICs) ranging from 0.25 to 2.5 mg/mL. The in vitro 3-(4,5-dimethyl-2-thizolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay determined that CS at 2000, 1000, 100, and 50 μg/mL could stimulate the proliferation of HPDLCs. Instead, HTCC inhibited the proliferation at the same concentrations but accelerated the proliferation of HPDLCs at relatively low concentrations (10, 3, 1.5, 1, and 0.3 μg/mL). Transmission electron microscopy (TEM) observations revealed that the ultra-architecture of HPDLC was seriously destroyed by HTCC treatment at 1000 μg/mL. Taken together, these results contribute information necessary to enhance our understanding of CS and HTCC in the dental field.     

Novel complex hydrogels based on N-carboxyethyl chitosan and quaternized chitosan and their controlled in vitro protein release property

A novel pH-responsive hydrogel (CHC) composed of N-carboxyethyl chitosan (CEC) and N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC) was synthesized by the redox polymerization technique. Turbidimetric titrations were used to determine the stoichiometric ratio of these two chitosan derivatives. The hydrogel was characterized by FT-IR, thermal gravimetric analysis (TGA), X-ray diffractometry (XRD), and scanning electron microscopy (SEM). The dynamic transport of water showed that the hydrogel reached equilibrium within 48 h. The swelling ratio of CHC hydrogel depended significantly on the pH of the buffer solution. The performance of the CHC as a matrix for the controlled release of BSA was investigated. It was found that the release behavior was determined by pH value of the medium as well as the intermolecular interaction between BSA and the hydrogels.     

Enhanced adsorption of congo red from aqueous solutions by chitosan hydrogel beads impregnated with cetyl trimethyl ammonium bromide

The adsorption of congo red (CR) onto chitosan (CS) beads impregnated by a cationic surfactant (CTAB, cetyl trimethyl ammonium bromide) was investigated. Chitosan beads impregnated at a ratio of 1/20 of CTAB to CS (0.05% of CTAB and 1% of CS) increased the CR adsorption capacity by 2.2 times from 162.3 mg/g (0% CTAB) to 352.5 mg/g (0.05% CTAB). The CR adsorption decreased with an increase in pH of the CR solution from 4.0 to 9.0. The Sips isotherm model showed a good fit with the equilibrium experimental data and the values of the heterogeneity factor (n) indicated heterogeneous adsorption of CR onto CS/CTAB beads, as well as CS beads. The kinetic data showed better fit to the pseudo second-order rate model than to the pseudo first-order rate model. The impregnation of CS beads by cationic surfactants showed the highest adsorption capacities of CR compared to any other adsorbents and would be a good method to increase adsorption efficiency for the removal of anionic dyes in a wastewater treatment process.     

Synthesis and antifungal properties of sulfanilamide derivatives of chitosan

Sulfanilamide derivatives of chitosan (2-(4-acetamido-2-sulfanimide)-chitosan (HSACS, LSACS), 2-(4-acetamido-2-sulfanimide)-6-sulfo-chitosan (HSACSS, LSACSS) and 2-(4-acetamido-2-sulfanimide)-6-carboxymethyl-chitosan (HSACMCS, LSACMCS)) were prepared using different molecular weights of chitosan (CS), carboxymethyl chitosan (CMCS) and chitosan sulfates (CSS) reacted with 4-acetamidobenzene sulfonyl chloride in dimethylsulfoxide solution. The structures of the derivatives were characterized by FT-IR spectroscopy and elemental analysis, which showed that the substitution degree of sulfanilamide group of HSACS, HSACSS, HSACMCS, LSACS, LSACSS and LSACMCS were 0.623, 0.492, 0.515, 0.576, 0.463 and 0.477, respectively. The solubility of the derivatives (pH<7.5) was higher than that of chitosan (pH<6.5). The antifungal activities of the derivatives against Aiternaria solani and Phomopsis asparagi were evaluated based on the method of Jasso et al. in the experiment. The results indicated that all the prepared sulfanilamide derivatives had a significant inhibiting effect on the investigated fungi in the polymer concentration range from 50 to 500 microg mL(-1). The antifungal activities of the derivatives increased with increasing the molecular weight, concentration or the substitution degree. The sulfanilamide derivatives of CS, CMCS and CSS show stronger antifungal activities than CS, CMCS and CSS.     

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.     

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.     

A comparative study on hypolipidemic activities of high and low molecular weight chitosan in rats

The hypolipidemic activities of high (712.6kDa) and low (39.8kDa) molecular weight chitosan (HMWC and LMWC) were evaluated in rats fed high-fat diets. Thirty-two male Sprague-Dawley rats in four groups were fed on three high-fat diets with each of them containing HMWC, LMWC or cellulose (high-fat control), and a control normal-fat diet for eight weeks. Compared with HMWC group, LMWC group showed decreased body weight gain, serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), as well as decreased liver triglyceride (TG). Fecal fat and cholesterol of LMWC group was lower than those of HMWC group. However, the activities of liver and serum lipoprotein lipase (LPL) of LMWC group were increased compared with HMWC group. The obtained results suggested that hypolipidemic activity of LMWC was better than HMWC, which might be partially attributed to the increase of serum and liver LPL activities.     

Physicochemical characterization of chitin and chitosan from crab shells

Crab chitosan was prepared by alkaline N-deacetylation of crab chitin for 60, 90 and 120 min and the yields were 30.0-32.2% with that of chitosan C120 being the highest. The degree of N-deacetylation of chitosans (83.3–93.3%) increased but the average molecular weight (483–526 kDa) decreased with the prolonged reaction time. Crab chitosans showed lower lightness and WI values than purified chitin, chitosans CC and CS but higher than crude chitin. With the prolonged reaction time, the nitrogen (8.9–9.5%), carbon (42.2–45.2%) and hydrogen contents (7.9–8.6%) in chitosans prepared consistently increased whereas N/C ratios remained the same (0.21). Crab chitosans prepared showed a melting endothermic peak at 152.3–159.2 °C. Three chitosans showed similar microfibrillar crystalline structure and two crystalline reflections at 2θ = 8.8–9.0° and 18.9–19.1°. Overall, the characteristics of three crab chitosans were unique and differed from those of chitosan CC and CS as evidenced by the element analysis, differential scanning calorimetry, scanning electron microscopy and X-ray diffraction patterns.     

Effect of the addition mode of carbon nanotubes for the production of chitosan hydrogel core-shell beads on adsorption of Congo red from aqueous solution

The adsorption performance of chitosan (CS) hydrogel beads (CSBs) generated by sodium dodecyl sulfate (SDS) gelation with multi-walled carbon nanotube (CNT) impregnation was investigated for Congo red removal as a model anionic dye. CNT-impregnated CSBs were prepared by four different strategies for dispersing CNTs: (a) in CS solution (CSBN1), (b) in SDS solution (CSBN2), (c) in CS solution containing cetyltrimethylammonium bromide (CTAB) (CSBN3), and (d) in SDS solution for gelation with CTAB-containing CS solution (CSBN4). It was observed from FE-SEM study that depending on nature of CNT dispersion, CNTs were found on the outer surface of CSBN2 and CSBN4 only. The adsorption capacity of the CSBs varied with the strategy used for CNT impregnation, and CSBN4 exhibited the highest maximum adsorption capacity (375.94 mg/g) from the Sips model. The lowest Sips maximum adsorption capacity by CSBN3 (121.07 mg/g) suggested significant blocking of binding sites of CS by CNT impregnation.     

Congo red adsorption from aqueous solutions by using chitosan hydrogel beads impregnated with nonionic or anionic surfactant

The adsorption performance of CS beads impregnated with triton X-100 (TX-100) as a nonionic surfactant and sodium dodecyl sulfate (SDS) as an anionic surfactant was investigated for the removal of anionic dye (congo red) from aqueous solution. While the adsorption capacity of CS/TX-100 beads was enhanced at all concentrations of TX-100 (0.005–0.1%), the increase in the concentration of SDS above 0.01% in the CS/SDS beads gradually reduced the adsorption capacity of the beads. Equilibrium adsorption isotherm data indicated a good fit to the Sips isotherm model and a heterogeneous adsorption process. The Sips maximum adsorption capacity in dry weight of the CS/TX-100 beads was 378.79 mg/g and 318.47 mg/g for the CS/SDS beads, higher than the 223.25 mg/g of the CS beads. Modification of CS beads by impregnation with nonionic surfactant, or even anionic surfactant, at low concentrations is a possible way to enhance adsorption of anionic dye.     

Preparation of low molecular weight chitosan using solution plasma system

The solution plasma system was introduced to treat chitosan solution in order to prepare low molecular weight chitosan. The plasma treatment time was varied from 0 min to 300 min. The plasma-treated chitosan was characterized including viscosity, molecular weight by GPC, and chemical characteristics by FT-IR. The results showed that after treated with plasma for 15-60 min, the viscosity of chitosan solution and apparent molecular weight of chitosans were remarkably decreased, compared to those of untreated sample. Longer treatment time had less effect on both viscosity and molecular weight of samples. Eventually, long treatment time (≥180 min) showed no influence on both viscosity and apparent molecular weight. This suggested that the degradation process of chitosan occurred during plasma treatment. FT-IR analysis revealed that chemical structure of chitosan was not affected by solution plasma treatment. TOF-MS results showed that chitooligosaccharides with the degree of polymerization of 2-8 were also generated by solution plasma treatment. The results suggested that solution plasma system could be a potential method for the preparation of low molecular weight chitosan and chitooligosaccharides.     

Amino acid modified chitosan beads: Improved polymer supports for immobilization of lipase from Candida rugosa

Amino acid modified chitosan beads (CBs) for immobilization of lipases from Candida rugosa were prepared by activation of a chitosan backbone with epichlorohydrin followed by amino acid coupling. The beads were analyzed by elemental analysis and solid state NMR with coupling yields of the amino acids ranging from 15 to 60%. The immobilized lipase on unmodified chitosan beads showed the highest immobilization yield (92.7%), but its activity was relatively low (10.4%). However, in spite of low immobilization yields (15–50%), the immobilized lipases on the amino acid modified chitosan beads showed activities higher than that of the unmodified chitosan beads, especially on Ala or Leu modified chitosan beads (Ala-CB or Leu-CB) with 49% activity for Ala-CB and 51% for Leu-CB. The immobilized lipases on Ala-CB improved thermal stability at 55 °C, compared to free and immobilized lipases on unmodified chitosan beads and the immobilized lipase on Ala-CB retained 93% of the initial activity when stored at 4 °C for 4 weeks. In addition, the activity of the immobilized lipase on Ala-CB retained 77% of its high initial activity after 10 times of reuse. The kinetic data (k_cat/K_m) supports that the immobilized lipase on Ala-CB can give better substrate specificity than the unmodified chitosan beads.     

Cytotoxicity and genotoxicity of chitooligosaccharides upon lymphocytes

Two COS mixtures and a low molecular weight chitosan (LMWC) were tested for potential cytotoxicity and genotoxicity upon human lymphocytes. Genotoxicity was evaluated in vitro by cytokinesis-blocked micronucleus and alkaline comet assays, while cytotoxicity was assessed by flow cytometry analysis. Our results suggest that COS do not exhibit any genotoxicity upon human lymphocytes, independently of MW or concentration. However, above 0.07 mg/mL COS induced strong cytotoxic effects. According to the concentration used, such cytotoxicity will induce cell death, essentially by necrosis (>0.10 mg/mL) and/or apoptosis (<0.10 mg/mL). The level of necrosis/apoptosis induced by high COS concentrations, suggests a promising use as apoptosis inducers in specific cancer situations.     

Chitosan derivatives alter release profiles of model compounds from calcium phosphate implants

The aim of the current study was to evaluate the impact of chitosan derivatives, namely N-octyl-chitosan and N-octyl-O-sulfate chitosan, incorporated in calcium phosphate implants to the release profiles of model drugs. The rate and extent of calcein (on M.W. 650 Da) ED, and FITC-dextran (M.W. 40 kDa) on in vitro release were monitored by fluorescence spectroscopy. Results show that calcein release is affected by the type of chitosan derivative used. A higher percentage of model drug was released when the hydrophilic polymer N-octyl-sulfated chitosan was present in the tablets compared with the tablets containing the hydrophobic polymer N-octyl-chitosan. The release profiles of calcein or FD from tablets containing N-octyl-O-sulfate revealed a complete release for FD after 120 h compared with calcein where 20% of the drug was released over the same time period. These results suggest that the difference in the release profiles observed from the implants is dependent on the molecular weight of the model drugs. These data indicate the potential of chitosan derivatives in controlling the release profile of active compounds from calcium phosphate implants.     

Improved stability and catalytic activity of graphene oxide/chitosan hybrid beads loaded with porcine liver esterase

Graphene oxide/chitosan and reduced graphene oxide/chitosan (GO/CS and RGO/CS) beads were prepared by precipitation with NaOH. Porcine liver esterase was immobilized on these beads to give GO/CS/E and RGO/CS/E beads. The optimum conditions for the maximum activity of RGO/CS/E beads were pH 8 and 50°C. The stability of the enzyme immobilized on GO/CS/E and RGO/CS/E was high in the pH range of 5–8. The GO/CS/E beads showed superior stability compared to that of the free enzyme and CS/E beads between 20 and 50°C. Kinetic analysis showed that GO/CS/E was a better catalyst than the RGO/CS/E beads with a lower K_m value of 0.9?mM. The hybrid beads also retained more than 95% activity after 10 consecutive cycles. The GO/CS/E and RGO/CS/E beads retained 84% and 87% activity after 40 days at 4°C. The GO/CS/E beads were used for the successful hydrolysis of methyl 4-hydroxy benzoate.