The impact of chitooligosaccharides and their derivatives on the in vitro and in vivo antitumor activity: A comprehensive review.

Chitooligosaccharides (COS) are the degraded products of chitin or chitosan. COS is water-soluble, non-cytotoxic to organisms, readily absorbed through the intestine, and eliminated primarily through the kidneys. COS possess a wide range of biological activities, including immunomodulation, cholesterol-lowering, and antitumor activity. Although work on COS goes back at least forty years, several aspects remain unclear. This review narrates the recent developments in COS antitumor activities, while paying considerable attention to the impacts of physicochemical properties (such as molecular weight and degrees of deacetylation) and chemical modifications both in vitro and in vivo. COS derivatives not only improve some physicochemical properties, but also expand the range of applications in drug and gene delivery. COS (itself or as a drug carrier) can inhibit tumor cell proliferation and metastasis, which might be attributed to its ability to stimulate the immune response along with its anti-angiogenic activity. Further, an attempt has been made to report limitations and future research. The potential health benefits of COS and its derivatives against cancer may offer a new insight on their applications in food and medical fields.

Pectin/lignocellulose nanofibers/chitin nanofibers bionanocomposite as an efficient biosorbent of cholesterol and bile salts.

A new biosorbent Ca-crosslinked pectin/lignocellulose nanofibers/chitin nanofibers (PLCN) was synthesized for cholesterol and bile salts adsorption from simulated intestinal fluid during gastric-intestinal passage. The physico-chemical properties of PLCN were studied using SEM, FTIR, XRD, DSC and BET. Before gastrointestinal passage, PLCN had an amorphous single-phase, compact structure formed via hydrogen and van der Waals bonds that revealed an irregular shape with the shriveled surface but watery condition and enzymatic digestion led to create a porous structure without destruction because of the water-insoluble nanofibers, therefore increasing the adsorption capacity. The maximum adsorption capacity reached 37.9 and 5578.4 mg/g for cholesterol and bile salts, respectively. Freundlich isotherm model indicated the reversible heterogeneous adsorption of both cholesterol and bile salts on PLCN. Further, their adsorption followed pseudo-second order kinetic model. These results suggest that PLCN has potential as a gastrointestinal-resistant biosorbent for cholesterol and bile salts adsorption applicable in medicine and food industry.

Antimicrobial Activity of Chitosan Oligosaccharides with Special Attention to Antiparasitic Potential.

The global rise of infectious disease outbreaks and the progression of microbial resistance reinforce the importance of researching new biomolecules. Obtained from the hydrolysis of chitosan, chitooligosaccharides (COSs) have demonstrated several biological properties, including antimicrobial, and greater advantage over chitosan due to their higher solubility and lower viscosity. Despite the evidence of the biotechnological potential of COSs, their effects on trypanosomatids are still scarce. The objectives of this study were the enzymatic production, characterization, and in vitro evaluation of the cytotoxic, antibacterial, antifungal, and antiparasitic effects of COSs. NMR and mass spectrometry analyses indicated the presence of a mixture with 81% deacetylated COS and acetylated hexamers. COSs demonstrated no evidence of cytotoxicity upon 2 mg/mL. In addition, COSs showed interesting activity against bacteria and yeasts and a time-dependent parasitic inhibition. Scanning electron microscopy images indicated a parasite aggregation ability of COSs. Thus, the broad biological effect of COSs makes them a promising molecule for the biomedical industry.

Recent Updates in Pharmacological Properties of Chitooligosaccharides.

Chemical structures derived from marine foods are highly diverse and pharmacologically promising. In particular, chitooligosaccharides (COS) present a safe pharmacokinetic profile and a great source of new bioactive polymers. This review describes the antioxidant, anti-inflammatory, and antidiabetic properties of COS from recent publications. Thus, COS constitute an effective agent against oxidative stress, cellular damage, and inflammatory pathogenesis. The mechanisms of action and targeted therapeutic pathways of COS are summarized and discussed. COS may act as antioxidants via their radical scavenging activity and by decreasing oxidative stress markers. The mechanism of COS antidiabetic effect is characterized by an acceleration of pancreatic islets proliferation, an increase in insulin secretion and sensitivity, a reduction of postprandial glucose, and an improvement of glucose uptake. COS upregulate the GLUT2 and inhibit digestive enzyme and glucose transporters. Furthermore, they resulted in reduction of gluconeogenesis and promotion of glucose conversion. On the other hand, the COS decrease inflammatory mediators, suppress the activation of NF-κB, increase the phosphorylation of kinase, and stimulate the proliferation of lymphocytes. Overall, this review brings evidence from experimental data about protective effect of COS.

Linolenic acid-modified methoxy poly (ethylene glycol)-oligochitosan conjugate micelles for encapsulation of amphotericin B.

Introduction of linolenic acid (LNA) and methoxy poly (ethylene glycol) (MPEG) to the backbone of oligochitosan (CS) afforded LNA-modified MPEG-CS conjugate (MPEG-CS-LNA). Amphotericin B-loaded MPEG-CS-LNA micelles (AmB-M) were prepared via dialysis method with 82.27 ± 1.96% of drug encapsulation efficiency and 10.52 ± 0.22% of drug loading capacity. The AmB-M enhanced AmB's water-solubility to 1.64 mg/mL, being 1640-folds higher than native AmB. The AmB-M obviously reduced hemolytic effect and renal toxicity of AmB when compared to marketed AmB injection (AmB-I). Its antifungal activity against Candida albicans was equivalent to AmB-I although AmB's release from AmB-M was significantly retarded. According to fluorescence microscopy test, the unchanged activity should be attributed to enhanced fungal cellular uptake of AmB-M caused by combined inducement of LNA and CS. The pharmacokinetic studies demonstrated that AmB-M also improved the pharmacokinetic parameters of AmB with AmB-I as control. Conclusively, developed LNA-modified MPEG-CS micellar system could be a viable alternative to the current toxic commercial AmB-I as a highly efficacious drug delivery system.

In vivo anti-psoriatic activity, biodistribution, sub-acute and sub-chronic toxicity studies of orally administered methotrexate loaded chitin nanogel in comparison with methotrexate tablet.

The anti-psoriatic efficacy of orally administered methotrexate loaded chitin nanogel (MCNG) was evaluated (two doses- 2.715 mg/kg and 5.143 mg/kg) and compared against orally administered methotrexate tablet MTX (5.143 mg/kg). MCNG at both dose levels of 2.715 mg/kg and 5.143 mg/kg exhibited significant anti-psoriatic activity which is very much comparable with MTX, caused normalization of histological features and inflammatory score associated with induced psoriasis. Biodistribution studies revealed the presence of drug in serum and in vital organs at all the three cases with highest amount in MCNG at 5.143 mg/kg dose, followed by MTX tablet and are lowest in MCNG at 2.715 mg/kg dose. MCNG at the highest dose of 5.143 mg/kg caused liver, lung and kidney toxicities on sub acute toxicity studies and MTX tablet was found to be toxic on liver and lung on sub chronic toxicity studies. MCNG 2.715 mg/kg was found to be safe on both sub acute and sub chronic administrations, suggesting that it can provide sufficient serum and tissue level of methotrexate necessary to clear psoriatic lesions, without inducing systemic toxicity and expected to be a better alternative for orally administered conventional methotrexate tablet for patients who need systemic medications for psoriasis.

Curcumin loaded chitin-glucan quercetin conjugate: Synthesis, characterization, antioxidant, in vitro release study, and anticancer activity.

In this study, we have synthesized chitin-glucan quercetin conjugate (ChGCQ) by an easy and facile free radical grafting reaction. The structure of ChGCQ was confirmed by proton nuclear magnetic resonance (1H NMR) and Fourier transforms infrared spectroscopy (FT-IR). Curcumin was loaded into ChGCQ to study its anti-cancer efficiency. The biocompatibility of ChGCQ and curcumin loaded ChGCQ (Cu-ChGCQ) were analysed by different assays in Peripheral blood mononuclear cells (PBMCs) and cytotoxicity test was performed in a macrophage cancer cell line (J774). The result shows tremendous biocompatibility of ChGCQ and Cu-ChGCQ in peripheral blood mononuclear cells and excellent cytotoxity in macrophage cancer cell line (J774). Chitin-glucan complex (ChGC), ChGCQ and Cu-ChGCQ showed 51%, 66% and 74% of DPPH radical-scavenging activity at 1mg/ml respectively, which are much higher than that of ChGC and in ABTS*+ assay 58%, 71% and 83% show radical-scavenging activity at 1mg/ml. Antioxidant assay of Cu-ChGCQ conjugate expressed much higher antioxidant activity than ChGCQ and ChGC. In vitro drug release study of Cu-ChGCQ conjuagate showed faster drug release in acidic medium in comparison to PBS of physiological pH and anticancer activity in vitro assay showed more anticancer activity of Cu-ChGCQ in comparison to ChGCQ conjugate.

Ultrasound stimulated release of gallic acid from chitin hydrogel matrix.

Ultrasound (US) stimulated drug release was examined in this study using a chitin hydrogel matrix loaded with gallic acid (GA), a drug used for wound healing and anticancer. Using phase inversion, GA-chitin hydrogels were prepared from chitin-dimethylacetamide (DMAc)/lithium chloride (LiCl) solution in the presence of GA, with 24h exposure of the solution to water vapor. The GA release from the GA-chitin hydrogel was examined under different US powers of 0-30W at 43kHz. The effects of GA loading amounts in the hydrogels (0.54, 0.43, and 0.25mg/cm3) and chitin concentrations (0.1, 0.5, and 1wt%) on the release behaviors were recorded under 43kHz US exposure at 30W. Results show that US accelerated the release efficiencies for all samples. Furthermore, the release efficiency increased concomitantly with increasing US power, GA loading amount, and decrease of the chitin concentration. The highest release rate of 0.74µg/mL·min was obtained from 0.54mg/cm3 of GA-loaded hydrogel fabricated from a 0.1wt% chitin mixture solution under 43kHz US exposure at 30W: nine times higher than that of the sample without US exposure. The hydrogel viscoelasticity demonstrated that the US irradiation rigidified the material. FT-IR showed that US can break the hydrogen bonds in the GA-chitin hydrogels.

Synthesis, biodistribution and excretion of radiolabeled poly(2-alkyl-2-oxazoline)s.

Here we report on the preparation of well defined water-soluble poly(2-methyl-2-oxazoline) and poly(2-ethyl-2-oxazoline) terminally equipped with a chelator (N,N',N'',N'''-tetraazacylododecane-1,4,7,10-tetraacetic acid (DOTA)) for radionuclide labeling. The tissue distribution and excretion of (111)In-labeled poly(2-alkyl-2-oxazoline)s were studied in mice. We found that the hydrophilic polymers do not accumulate in tissues and are rapidly cleared from the blood pool, predominantly by glomerular filtration in the kidneys. In contrast only a small fraction is excreted via the hepatobiliary tract. Only minimal amounts of poly(2-alkyl-2-oxazoline)s are taken up by the reticuloendothelial system (RES). Scintigraphic studies revealed the feasibility of in vivo imaging of (111)In-labeled poly(2-oxazoline)s. Since additional functionalities for targeting can readily be introduced into poly(2-oxazoline)s via functional monomer units, these compounds fulfill fundamental requirements for an application as carrier molecules in radionuclide therapy.

Thiolated chitosan microparticles: a vehicle for nasal peptide drug delivery.

The goal of this study was to develop a microparticulate delivery system based on a thiolated chitosan conjugate for the nasal application of peptides. Insulin was used as model peptide. For thiolation of chitosan 2-iminothiolane was covalently linked to chitosan. The resulting chitosan-TBA (chitosan-4-thiobutylamidine) conjugate featured 304.89+/-63.45 micromol thiol groups per gram polymer. 6.5% of these thiol groups were oxidised. A mixture of the chitosan-TBA conjugate, insulin and the permeation mediator reduced glutathione were formulated to microparticles. Control microparticles comprised unmodified chitosan and insulin. As second control served mannitol-insulin microparticles. All microparticulate systems were prepared via the emulsification solvent evaporation technique. In 100 mM phosphate buffer pH 6.8 chitosan-TBA-insulin microparticles swelled 4.39+/-0.52-fold in size, whereas chitosan based microparticles did not swell at all. Chitosan-TBA microparticles showed a controlled release of fluorescein isothiocyanate (FITC)-labelled insulin over 6 h. Nasal administered chitosan-TBA-insulin microparticles led to an absolute bioavailability of 7.24+/-0.76% (means+/-S.D.; n=3) in conscious rats. In contrast, chitosan-insulin microparticles and mannitol-insulin microparticles exhibited an absolute bioavailability of 2.04+/-1.33% and 1.04+/-0.27%, respectively (means+/-S.D.; n=4). Because of these results microparticles comprising chitosan-TBA and reduced glutathione seem to represent a useful formulation for the nasal administration of peptides.

Biodistribution and excretion of radioactivity after the administration of 166Ho-chitosan complex (DW-166HC) into the prostate of rat.

PURPOSE: The objective of this study was to determine the fate of the 166Ho-chitosan complex (DW-166HC) in rats by examining its absorption, distribution and excretion after administration into the prostate. METHODS: About 100 microCi of DW-166HC [containing 0.1875 mg of Ho(NO3)3.5H2O and 0.25 mg of chitosan] was administered intraprostatically. The level of radioactivity in blood, urinary and faecal excretion, and radioactivity distribution were examined. To determine the effect of chitosan in DW-166HC, 166Ho nitrate alone [0.1875 mg of Ho(NO3)3.5H2O] was administered into the prostate of male rats, and radioactivity distribution was examined using whole-body autoradiography. RESULTS: After administration of DW-166HC into the prostate, cumulative urinary and faecal excretion over the period 0-72 h was 0.35% and 0.11%, respectively. The radioactivity at the administration site was extremely high at all time points up to 144 h (>98% of injected dose). The small amount of radioactivity which did transfer from the administration site distributed mainly to the liver, spleen, kidney cortex and bone. Compared with the DW-166HC group, the group that received 166Ho nitrate alone displayed three- to fourfold higher levels of radioactivity in the main tissues, including liver, spleen, kidney cortex and bone, at 24 h after administration (P < 0.05). CONCLUSION: The results of this study show clearly that most of the administered DW-166HC remained at the administration site. It is concluded that the chitosan complex may be used to retain 166Ho within a limited area in cancer of the prostate.

Contribution of chitosan and its derivatives to cancer chemotherapy.

The conjugates of some kinds of anticancer agents with chitin and chitosan derivatives display good anticancer effects with a decrease in the adverse effects of the original drug due to a predominant distribution into the cancer and a gradual release of free drug from the conjugates. For instance, doxifluridine and 1-beta-D-arabinofuranosylcytosine (Ara-C) were conjugated with chitosan via glutaric spacer, and the conjugates of Ara-C with chitosan, in particular, showed a good antitumour effect against P388-bearing leukemia model mice. Glycol-chitosan (G-Chi) was distributed mainly in the systemic circulation and the kidney after i.v. administration into normal mice, and retained long in the kidney. The therapeutic effect of the conjugates of mitomycin C (MMC) with G-Chi was not necessarily improved in comparison with that of the free drug, but toxic side-effects appeared to decrease with the conjugates. The conjugates of MMC with 6-O-carboxymethyl-chitin showed almost complete suppression of tumour growth at 10 mg eq. MMC/kg, though a lethal adverse effect was also observed. The conjugates of MMC with N-succinyl-chitosan showed good antitumour activities against various tumour models due to their predominant distribution into the tumour tissue and sustained-release characteristics, irrespective of water-insoluble and -soluble formulations. It is believed that the chitin and chitosan derivatives discussed in this review are good candidates for a polymeric drug carrier in cancer chemotherapy.

[Research on carboxymethyl chitosan acting as the adjuvant for implantable degradable microspheres].

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.

Chitosan nanoparticles for plasmid DNA delivery: effect of chitosan molecular structure on formulation and release characteristics.

Chitosan can be useful as a nonviral vector for gene delivery. Although there are several reports to form chitosan-pDNA particles, the optimization and effect on transfection remain insufficient. The chitosan-pDNA nanoparticles were formulated using complex coacervation and solvent evaporation techniques. The important parameters for the encapsulation efficiency were investigated, including molecular weight and deacetylation degree of chitosan. We found that encapsulation efficiency of pDNA is directly proportional with deacetylation degree, but there is an inverse proportion with molecular weight of chitosan. DNA-nanoparticles in the size range of 450-820 nm depend on the formulation process. The surface charge of the nanoparticles prepared with complex coacervation method was slightly positive with a zeta potential of +9 to +18 mV; nevertheless, nanoparticles prepared with solvent evaporation method had a zeta potential approximately +30 mV. The pDNA-chitosan nanoparticles prepared by using high deacetylation degree chitosan having 92.7%, 98.0%, and 90.4% encapsulation efficiency protect the encapsulated pDNA from nuclease degradation as shown by electrophoretic mobility analysis. The release of pDNA from the formulation prepared by complex coacervation was completed in 24 hr whereas the formulation prepared by evaporation technique released pDNA in 96 hr, but these release profiles are not statistically significant compared with formulations with similar structure (p > .05). According to the results, we suggest nanoparticles have the potential to be used as a transfer vector in further studies.

Controlled release of Bordetella bronchiseptica dermonecrotoxin (BBD) vaccine from BBD-loaded chitosan microspheres in vitro.

Chitosan microspheres were prepared by ionic gelation process with sodium sulfate for nasal vaccine delivery. Bordetella Bronchiseptica Dermonecrotoxin (BBD) as a major virulence factor of a causative agent of atrophic rhinitis (AR) was loaded to the chitosan microspheres for vaccination. Morphology of BBD-loaded chitosan microspheres was observed as spherical shapes. The average particle sizes of the BBD-loaded chitosan microspheres were about 2.69 microm. More BBD was released with an increase of molecular weight of chitosan and with an increase of medium pH in vitro due to weaker intermolecular interaction between chitosan and BBD. Tumor necrosis factor-alpha (TNFalpha) and nitric oxide (NO) from RAW264.7 cells stimulated with BBD-loaded chitosan microspheres were gradually secreted, suggesting that released BBD from chitosan microspheres had immune stimulating activity of AR vaccine.

Receptor-mediated stimulatory effect of oligochitosan in macrophages.

Oligochitosan, which has greater than 3 but less than 10 saccharide (N-acetylglucosamine or glucosamine) residues, is obtained by either chemical or enzymatic hydrolysis of chitosan. In this work, we demonstrated that oligochitosan had an in vitro stimulatory effect on the release of tumor necrosis factor-alpha and interleukin-1 beta in macrophages. Moreover, we observed that oligochitosan could be uptaken by macrophages through confocal laser microscopy. Scatchard analysis of internalization of 2-aminoacridone-oligochitosan in macrophages indicated its internalization was mediated by a specific receptor on macrophage membrane with a Kd of 2.1 x 10(-5) M. Competition studies showed that mannose could inhibit oligochitosan internalization, while lipopolysaccharide and beta-glucan could not do it. Inhibition of mannose-BSA, fucose-BSA, and N-acetylglucosamine-BSA on oligochitosan internalization further suggests that oligochitosan internalization is mediated by a macrophage lectin receptor like with mannose specificity.

Anti-ulcerogenic effect of chitin and chitosan on mucosal antioxidant defence system in HCl-ethanol-induced ulcer in rats.

The anti-ulcerogenic effect of chitin and chitosan against ulcer induced by HCl-ethanol in male Wistar rats was studied. Levels of acid output, pepsin, protein, lipid peroxides and reduced glutathione and the activity of glutathione peroxidase (GPx), glutathione-S-transferase (GST), catalase (CAT) and superoxide dismutase (SOD) were determined in the gastric mucosa of normal and experimental groups of rats. A significant increase in volume and acidity of the gastric juice was observed in the ulcer-induced group of rats. Peptic activity was significantly decreased as compared with that of normal controls. In the rats pre-treated with chitin and chitosan 2% along with feed, the volume and acid output and peptic activity of gastric mucosa were maintained at near normal levels. The level of lipid peroxidation was significantly higher in the ulcerated mucosa when compared with that of normal controls. This was paralleled by a decline in the level of reduced glutathione and in the activity of antioxidant enzymes like GPx, GST, CAT and SOD in the gastric mucosa of ulcer-induced rats. Also, the levels of mucosal proteins and glycoprotein components were significantly depleted in ulcerated mucosa. The pre-treatment with chitin and chitosan was found to exert a significant anti-ulcer effect by preventing all the HCl-ethanol-induced ulcerogenic effects in experimental rats.

Chemotherapeutic potential of alginate-chitosan microspheres as anti-tubercular drug carriers.

OBJECTIVES: This study was designed to develop alginate-chitosan microspheres as drug carriers to reduce dose/dosing frequency in the management of tuberculosis (TB), which otherwise demands prolonged chemotherapy. METHODS: Alginate-chitosan microspheres encapsulating three frontline anti-tuberculous drugs (ATDs), rifampicin, isoniazid and pyrazinamide, were formulated. A therapeutic dose and a half-therapeutic dose of the microsphere-encapsulated ATDs were orally administered to guinea pigs for pharmacokinetic/chemotherapeutic evaluations, respectively. RESULTS: The drug encapsulation efficiency ranged from 65% to 85% with a loading of 220-280 mg of drug per gram microspheres. Administration of a single oral dose of the microspheres to guinea pigs resulted in sustained drug levels in the plasma for 7 days and in the organs for 9 days. The half-life and mean residence time of the drugs were increased 13- to 15-fold by microsphere encapsulation, along with an enhanced relative/absolute bioavailability. The sustained release and increase in bioavailability were also observed with a sub-therapeutic dose of the microspheres. In Mycobacterium tuberculosis H37Rv-infected guinea pigs, administration of a therapeutic dose of microspheres spaced 10 days apart produced a clearance of bacilli equivalent to conventional treatment for 6 weeks. The most important observation, however, was the documentation of therapeutic benefit with a half-therapeutic dose of the microspheres administered weekly. CONCLUSION: Alginate-chitosan microspheres hold promise as a potential natural polymer-based oral ATD carrier for better management of TB.

Preparation of alginate/chitosan microcapsules and enteric coated granules of mistletoe lectin.

The aqueous extract of European mistletoe (Viscum album, L.) has been used in cancer therapy. The purified mistletoe lectins, main components of mistletoe, have demonstrated cytotoxic and immune-system-stimulating activities. Korean mistletoe (Viscum album L. coloratum), a subspecies of European mistletoe, has also been reported to possess anticancer and immunological activities. A galactose- and N-acetyl-D-galactosamine-specific lectin (Viscum album L. coloratum agglutinin, VCA) with Mr 60 kDa was isolated from Korean mistletoe. Mistletoe preparations have been given subcutaneously due to the low stability of lectin in the gastrointestinal (GI) tract. In the present study, we investigated the possibility of alginate/chitosan microcapsules as a tool for oral delivery of mistletoe lectin. In addition, our strategy has been to develop a system composed of stabilizing cores (granules), which contain mistletoe lectin, extract or powder, coated by a biodegradable polymer wall. Our results indicated that successful incorporation of VCA into alginate/chitosan microcapsules has been achieved and that the alginate/chitosan microcapsule protected the VCA from degradation at acidic pH values. And coating the VCA with polyacrylic polymers, Eudragit, produced outstanding results with ideal release profiles and only minimal losses of cytotoxicity after manufacturing step. The granules prepared with extract or whole plant produced the best results due to the stability in the extract or whole plant during manufacturing process.

Effect of chitosan on epithelial cell tight junctions.

PURPOSE: Chitosan has been proposed as a novel excipient for transepithelial drug-delivery systems. Chitosan is thought to disrupt intercellular tight junctions, thus increasing the permeability of an epithelium. The effect of chitosan on tight junction complex was investigated at the molecular level. METHODS: Changes in barrier properties of Caco-2 cell monolayers, including transepithelial electrical resistance and permeability to horseradish peroxidase (HRP), were assessed in response to chitosan treatment. Changes in subcellular localization of the tight junction proteins zona occludens 1 (ZO-1) and occludin by immunofluorescence and Western blotting of cellular fractions were also assessed. RESULTS: Chitosan was found to cause a dose-dependent reduction in transepithelial electrical resistance of Caco-2 monolayers of up to 83%. A corresponding increase in horseradish peroxidase permeability of up to 18 times greater than the control was also observed across the monolayer. Immunofluorescent localization of ZO-1 revealed loss of membrane-associated ZO-1 from discrete areas. Analysis of cellular fractions revealed a dose-dependent loss of ZO-1 and occludin from the cytosolic and membrane fractions into the cytoskeletal fraction. These changes did not occur because of chitosan-mediated ATP depletion. CONCLUSIONS: Chitosan-mediated tight junction disruption is caused by a translocation of tight junction proteins from the membrane to the cytoskeleton.