Chitinase has a wide industrial application prospect. For example, it can degrade shrimp shells, crab shells and other crustacean waste into high value-added chitooligosaccharides. However, the low catalytic efficiency of chitinase greatly limits the production of chitooligosaccharides. In previous study, the we expressed a chitinase Chisb with high catalytic efficiency and studied its enzymatic properties. In order to further improve the catalytic efficiency of Chisb, with R13NprB-C-SP-H as the parent, here error-prone PCR was used to construct random mutant library to conduct directed evolution of chitinase Chisb. Two mutants C43D and E336R were obtained with 96-well plate primary screening and shaker-screening, and their enzymatic properties were also studied. The optimum temperature of C43D and E336R was 55 °C, and the optimum pH of C43D was 5.0, while that of E336R was 9.0. The catalytic efficiency of C43D and E336R was 1.35 times and 1.57 times higher than that of control. The chitooligosaccharide concentration of E336R and C43D was 2.53 g/L and 2.06 g/L, improved by 2.84 times and 2.31 times compared with the control (0.89 g/L), respectively. In addition, the substrate conversion rate of mutants E336R and C43D was 84.3% and 68.7%, improved by 54.6% and 39% compared with the control (29.7%), respectively. In summary, the study indicates that random mutation introduced by error-prone PCR can effectively improve the catalytic efficiency of chitinase Chisb. The positive mutants with higher catalytic efficiency obtained in the above study and their enzymatic property analysis have important research significance and application value for the biosynthesis of chitooligosaccharides.
Biocatalysis ; Chitin ; analogs & derivatives ; Chitinases ; Hydrogen-Ion Concentration ; Polymerase Chain Reaction

OBJECTIVETo investigate the effect of oligochitosan in promoting intestinal absorption of protoberberine alkaloids in extracts from Corydalis saxicola total alkaloids.

METHODThe in vitro single-pass intestinal perfusion model in rats was established to study the changes in absorption kinetic parameters of dehydrocavidine, berberine hydrochloride and palmatine chloride in C. saxicola total alkaloids after the addition of different concentrations oligochitosan and evaluate the effect of oligochitosan in promoting intestinal absorption of the drugs.

RESULTThe concentration of oligochitosan had different effects on the absorption rate constant (Ka) and apparent permeability coefficient (Peff) of the three active component in rat intestines. Ka and Peff in 0.5% oligochitosan group significantly increased, indicating a stronger effect in promoting the absorption.

CONCLUSIONOligochitosan has a certain effect in promoting the intestinal absorptions of protoberberine alkaloids in C. saxicola total alkaloids.

Animals ; Berberine Alkaloids ; administration & dosage ; pharmacokinetics ; Chitin ; administration & dosage ; analogs & derivatives ; Corydalis ; chemistry ; Drugs, Chinese Herbal ; administration & dosage ; pharmacokinetics ; Intestinal Absorption ; drug effects ; Intestines ; drug effects ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley

P-glycoprotein (P-gp) located in the apicalmembranes of intestinal absorptive cells is an energy-dependent efflux pump which can reduce the bioavailability of a wide range of substrate drugs. There is increasingly interest in enhancing the bioavailability of these molecules by inhibiting intestinal P-gp. A classification of excipient inhibitors of intestinal P-gp nonionic surfactants, poly (ethylene glycol), derivates of beta-cyclodextrin and thiolated chitosan will be presented and then the inhibition mechanism will be discussed. Compared with traditional P-gp inhibitor, excipient inhibitors appear to have minimal nonspecific pharmacological activity, thus potential side effects can be mostly avoided. These excipient inhibitors, which hold the promise of replacing the traditional ones, will be extensively employed to significantly improve the intestinal absorption of poorly soluble and absorbed drugs as a result of P-gp inhibition, and thus to enhance the bioavailability of these drugs. However, the further studies of both the mechanism and clinical application are urgently needed.
ATP-Binding Cassette, Sub-Family B, Member 1 ; antagonists & inhibitors ; pharmacokinetics ; Animals ; Biological Availability ; Chitin ; analogs & derivatives ; pharmacology ; Excipients ; pharmacology ; Glycerol ; analogs & derivatives ; pharmacology ; Humans ; Intestinal Absorption ; drug effects ; Polyethylene Glycols ; pharmacology ; Surface-Active Agents ; pharmacology ; beta-Cyclodextrins ; pharmacology

Hydroxyethyl chitin (HECH) is a water soluble chitin derivative made by etherification of chitin, ethylene chlorohydrin was used as etherification reagent in this reaction. A novel interpenetrating polymer network (IPN) composed of HECH/PAA was prepared. The IR spectra confirmed that HECH/PAA was formed through chemical bond interaction. The sensitivity of this hydrogel to temperature and pH was studied. The swelling ratio of this hydrogel in artificial intestinal juice is much greater than that in artificial gastric juice. The IPN hydrogel exhibited a typical pH-sensitivity, and its degree of swelling ratio increased with the increase of temperature. The sustained-release drug system of Dichlofenac potassium was prepared by using HECH/PAA as the drug carrier. The release experiment showed a perfect release behavior in artificial intestinal juice. This IPN is expected to be used as a good drug delivery system of enteric medicine.
Acrylates ; administration & dosage ; chemistry ; Anti-Inflammatory Agents, Non-Steroidal ; administration & dosage ; Chitin ; administration & dosage ; analogs & derivatives ; chemistry ; Delayed-Action Preparations ; Diclofenac ; administration & dosage ; Drug Carriers ; chemical synthesis ; Drug Delivery Systems ; Hydrogel, Polyethylene Glycol Dimethacrylate ; administration & dosage ; chemistry ; Hydrogen-Ion Concentration

OBJECTIVETo study the mechanism of oligochitosan-induced macrophage activation.

METHODSOligochitosan was chemically modified with fluorophore 2-aminoacridone (2-AMAC). The cellular events of 2-AMAC-oligochitosan-macrophage interaction were analyzed with confocal laser microscopy and the fluorescence intensity of cells was analyzed by BD LSR flow cytometer. The mechanism of oligochitosan uptake by macrophages was studied by competitive inhibition test and the effect of calcium, trypsin and colchicine on oligochitosan recognition and internalization were also determined. RT-PCR was performed to investigate the level of TNF-alpha secretion.

RESULTMacrophage could bind and uptake oligochitosan, which was dependent on the temperature: the uptake proceeded rapidly at 37 degrees C and at 4 degrees C macrophage could only bind oligochitosan. EDTA decreased oligochitosan uptake. Trypsin treatment significantly reduced the internalization, and uptake was recovered by trypsin termination. Colchicine significantly inhibited the internalization process and was dose dependent. 0.1 mol/L mannose inhibited TNF-alpha expression induced by oligochitosan.

CONCLUSIONMacrophage could uptake oligochitosan via mannose receptor mediated pinocytosis. Mannose receptor is crucial for the oligochitosan-induced macrophages activation.

Cells, Cultured ; Chitin ; analogs & derivatives ; pharmacology ; Humans ; Lectins, C-Type ; metabolism ; Macrophage Activation ; drug effects ; Macrophages ; cytology ; Mannose-Binding Lectins ; metabolism ; Pinocytosis ; drug effects ; Receptors, Cell Surface ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo prepare microbubble, made of N-carboxymethyl chitosan, as ultrasound contrast agent and evaluate its characteristics and acoustic effects in vivo.

METHODSOil-Water-Oil multiple emulsion/solvent evaporation method was used to prepare the microbubble contrast agent. Both optical micrography and scanning electron micrography were performed to determine the bubble size and morphology. The acoustic effect of the N-carboxymethyl chitosan echo contrast agent was evaluated in vivo in rabbit. Liver echo images were recorded with ultrasound machine before and after intravenous bolus injecting 0.5 ml of the agent.

RESULTSThe novel N-carboxymethyl chitosan echo contrast agent was formulated as lyophilized product, with a mean diameter of 2-3 microm and a shell thickness of 250-300 nm. Its size is relatively uniform. The imaging effect was remarkably enhanced with the ultrasonic contrast agent when applied in rabbit livers.

CONCLUSIONIt is feasible to prepare excellent microbubble ultrasound contrast agent with N-carboxymethyl chitosan as membrane components.

Animals ; Chitin ; analogs & derivatives ; chemical synthesis ; chemistry ; Contrast Media ; Liver ; diagnostic imaging ; Microbubbles ; Rabbits ; Ultrasonics ; Ultrasonography

This study examined the effectiveness of Holmium-166 (Ho-166) chitosan complex therapy for a malignant glioma. Cultured C6 glioma cells (100, 000 in 5microliter) were injected into the caudate/putamen of 200 - 250 gram Wistar rats. Five days later, a Ho-166 chitosan complex was injected into the same site of the glioma injection. Four injection doses were administered: the control group received PBS 10microliter, group 1 received an injection of 100micro Ci (10microliter), group 2 received an injection of 50microCi (5microliter), and group 3 received an injection of 10micro Ci (1microliter). The average tumor volume for each group was 1.385 mm3 for the control group, 0.036 mm3 for group 1, 0.104 mm3 for group 2, and 0.111 mm3 for group 3. Compared with the control group, the size of the tumors in groups 1, 2 and 3 was reduced by an average of 97.4%, 92.5% and 91.9%, respectively. The Kaplan-Meier survival curve of group 2 was the longest, followed by groups 3, group 1 and the control. The mean survival was 22.8, 59, 60, and 44.6 days for the control group and groups 3, 2 and 1, respectively. H-E staining revealed that group 2 yielded the best results in the destruction of the malignant glioma. TUNEL staining and immunohistochemical studies indicated apoptotic features. The Ho-166 chitosan complex proved to be effective in destroying the malignant glioma.
Animals ; Brachytherapy ; Brain Neoplasms/mortality/pathology/*radionuclide imaging ; Cell Line, Tumor ; Chitin/analogs & derivatives/*pharmacology ; Disease Models, Animal ; Glioma/mortality/pathology/*radionuclide imaging ; Holmium/*pharmacology ; Radioisotopes/*pharmacology ; Rats ; Rats, Wistar ; Research Support, Non-U.S. Gov't

As a kind of biomaterial, carboxymethyl chitosan (CMC) has excellent biodegradable and bioacceptable capabilities using. This study was aimed to probe into the feasibility of CMC to prepare the implantable sustained release Ciprofloxacin Hydrochloride (CPX) microspheres(MS), and to go further into the pharmaceutic technology, the morphology and the characteristics of in vitro release of the microspheres. First, we prepared the microspheres by emulsification and cross-linking technology. Then, scanning electron microscopy (SEM), infrared spectrum (IR) and differential thermal analysis (DTA) were used to detect the structure and morphology of the MS. The in vitro release of CPX/CMC-MS and the CPX content of the MS were detected through continuous-flow releasing system. We found that the structure and morphology of the MS were affected by the conditions of preparation such as emulsification and cross-linking temperature, ionic strength and stirring speed, that the releasing time of CPX was more than 7 days, and that the releasing behaviors of the microspheres conformed to the Higuchi model. So we drew the conclusions that CMC could be used as a kind of absorbable and implantable adjuvant for sustained release, the technology of emulsification and cross-linking was proved to be feasible, stable and simple.
Absorbable Implants ; Biocompatible Materials ; Biodegradation, Environmental ; Chitin ; administration & dosage ; analogs & derivatives ; pharmacokinetics ; Chitosan ; Ciprofloxacin ; administration & dosage ; pharmacokinetics ; Cross-Linking Reagents ; Delayed-Action Preparations ; Drug Carriers ; chemical synthesis ; Humans ; Microspheres

We have prepared a wound dressing made from chitosan and collagen. Its clinical curative effect was detected. Chitosan solution was put into purified collagen solution. Then, the solution became sponge by means of freeze drying, and it was subjected to a series of toxicology tests, including acute toxicity, stimulation test, allergic and hemolysis tests, as well as the clinical test of openning trauma in orthopedics. All of the results of toxicology tests were negative. The chitosan-collagen sponge could not only accelerate the speed of curing but also restrain the extravasate. Therefore, the chitosan-collagen sponge has good biocompatibility and clinical curative effect. It is a prospective security-biomaterial for medical use.
Biocompatible Materials ; administration & dosage ; Biological Dressings ; Chitin ; analogs & derivatives ; isolation & purification ; therapeutic use ; Chitosan ; Collagen ; isolation & purification ; therapeutic use ; Humans ; Membranes, Artificial ; Wound Healing ; drug effects

Amphiphilic coupling-polymer of stearyl poly (ethylene oxide)-co-4, 4'-methylendiphenyl diisocyanate-co-stearyl poly(ethylene oxide), MSPEO, was specially designed as surface-modifying additives. The blends of MSPEO in both polyether urethane (PEU) and chitosan(Chi), as the coating materials for intravascular device were investigated. Two kinds of static clotting time tests, plasma recalcification time (PRT) and prothrombin time(PT), as well as the static platelet adhesion experiment were carried out. And the dynamic anti-coagulation experiment was performed with a closed-loop tubular system under a blood shear rate of 1,500 s-1. The results demonstrate that both blend coatings can improve the anti-coagulation of polyurethane greatly and will not lead to hemolysis, and that more platelets adhere to the surface modified by Chi-MSPEO blend coating as compared with those adhere to the surface modified by PEU-MSPEO blend coating. The surface modified by Chi-MSPEO has longer PRT, whereas the surface modified by PEU-MSPEO has longer PT.
Blood Coagulation Tests ; Chitin ; analogs & derivatives ; chemistry ; Chitosan ; Coated Materials, Biocompatible ; chemistry ; Ethylene Oxide ; chemistry ; Humans ; Materials Testing ; Platelet Adhesiveness ; Polymers ; chemistry ; Polyurethanes ; chemistry ; Prothrombin Time ; Surface Properties