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Chitosan (Chi) is a natural biodegradable cationic polymer with remarkable potency as a vehicle for drug or vaccine delivery. Chi possesses multiple groups, which can be used both for Chi derivatization and for particle formation. The aim of this work was to produce stable nanosized range Chi gels (nanogels, NGs) with different charge and to study the driving forces of complex formation between Chi NGs and proteins or peptides. Positively charged NGs of 150 nm in diameter were prepared from hexanoyl chitosan (HC) by the ionotropic gelation method while negatively charged NGs of 190 nm were obtained from succinoyl Chi (SC) by a Ca²⁺ coacervation approach. NGs were loaded with a panel of proteins or peptides with different weights and charges. We show that NGs preferentially formed complexes with oppositely charged molecules, especially peptides, as was demonstrated by gel-electrophoresis, confocal microscopy and HPLC. Complex formation was accompanied by a change in zeta-potential and decrease in size. We concluded that complex formation between Chi NGs and peptide/proteins is mediated mostly by electrostatic interactions.

Bovine herpesvirus 1 (BHV-1), is a major pathogen of cattle which causes serious infections, including infectious bovine rhinotracheitis (IBR)/infectious pustular vulvovaginitis (IPV). At present, BHV-1 is still a serious threat to animal health and productivity in Turkey, hence to develop a more efficient and economical vaccine system against BHV-1 is certainly an important necessity. A mucosal vaccination strategy would provide both mucosal and systemic immune responses to protect disease progression and transmission. However, vaccination through mucosal membranes requires adjuvants/delivery systems in order to enhance the immunogenicity of the antigens. Chitosan, which is a biodegradable, biocompatible and bioadhesive natural polysaccharide, has been shown to be promising both as a delivery system and an adjuvant for mucosal vaccination. In this study, microparticles with appropriate size (<10μm), positive surface charge and high loading efficiency (∼95%) were prepared for mucosal delivery of BHV-1, using various types of chitosan with different molecular weight and solubility. Particles were shown to be taken up by the cells, mostly around the nucleus, whereas with aggregates which were bigger in size were adsorbed at the surface. Furthermore, gel formulations with a suitable viscosity which would provide easy application and remain on the mucosa for extended period of time were also developed with a high zeta potential indicating a stable system. Both the BHV-1 loaded microparticle and gel formulations were shown to maintain cell viability and antigen integrity. Chitosan-based formulations are suggested as promising adjuvant/delivery systems for mucosal immunization against BHV-1., (Copyright © 2010 Elsevier B.V. All rights reserved.)

This study aimed to investigate and compare the effect of chitosan sponge and platelet-rich plasma (PRP) gel alone as well as their combination on bone regeneration in rabbit cranial defects. Four cranial defects with a 4.5-mm diameter were created in rabbit cranium and grafted with PRP, chitosan sponge alone, and chitosan sponge incorporated with PRP. The rabbits were killed by the fourth and eighth weeks, and the defects were analyzed histologically. Higher bone formation was observed in the PRP group when compared with the other groups at weeks 4 and 8. All parts of the defects were filled with thick trabecular new bone in the PRP group. The amount of new bone formation in the control groups was found to be less when compared with the PRP group and the least in the chitosan group. The defects that were filled with chitosan sponge showed a limited amount of new bone formation and an obvious fibrous demarcation line between chitosan particles and bone. Application of PRP showed a histological tendency toward increased bone formation. Other forms or derivatives of chitosan may have beneficial effects to achieve new bone regeneration.

In this study, for the first time, TMC/MCC complex nanoparticles as a delivery system and as an adjuvant were developed and evaluated to obtain systemic and mucosal immune responses against nasally administered tetanus toxoid (TT). Nanoparticles were developed by complexation between the oppositely charged chitosan derivatives, N-trimethyl chitosan (TMC, polycationic) and mono-N-carboxymethyl chitosan (MCC, polyampholytic) without using any crosslinker for mucosal vaccination. The cellular viability was found to be higher with TMC/MCC complex compared to that of MCC and TMC alone. Size, zeta potential and morphology of the nanoparticles were investigated as a function of preparation method. Nanoparticles with high loading efficacy (95%) and positively charged surface were obtained with an average particle size of 283+/-2.5 nm. The structural integrity of the TT in the nanoparticles was confirmed by SDS-PAGE electrophoresis analysis. Cellular uptake studies indicated that FITC-BSA loaded nanoparticles were effectively taken up into the mouse Balb/c monocyte macrophages. Mice were nasally immunized with TT loaded TMC/MCC complex nanoparticles and compared to that of TMC and MCC nanoparticles. TMC/MCC complex nanoparticles were shown to induce both the mucosal and systemic immune response indicating that this newly developed system has potential for mucosal administration of vaccines.

Periodontitis is a chronic infection in the supportive tissue of the teeth which eventually leads to tooth loss. Various grafting materials and barrier membranes have been used to repair periodontal intraosseous lesions. Chitosan is a derivative of chitin, a natural biopolymer, which is biologically safe, biodegradable, and nontoxic and has been applied in a variety of forms in dentistry. It also exerts bioactive properties such as wound healing, antimicrobial, tissue regeneration, and hemostatic activities. The aim of this study was to evaluate effects of chitosan on periodontal regeneration. Twenty chronic periodontitis patients were recruited. Following initial therapy, the patients were divided into four groups: group A, receiving chitosan gel (1% w/v); group B, receiving chitosan gel + demineralize bone matrix; group C: receiving chitosan gel + collagenous membrane; and group D, receiving flap only (control group). Clinical and radiographic measurements were recorded at baseline, day 90 (3rd month), and day 180 (6th month) after surgery. For clinical data, no significant differences were obtained among the treatment groups. However, radiographic data revealed that except control group, all the other groups showed statistically significant bone fills when compared with baseline indicating that chitosan gel alone or its combination with demineralize bone matrix/collagenous membrane is promising for periodontal regeneration., ((c) 2009 Wiley Periodicals, Inc.)

This review discusses the current status of chitosan and its derivatives as adjuvants and delivery systems in vaccine delivery, and their future possibilities and challenges. After a brief introduction to adjuvants and delivery systems, chitosan will be described in detail in regard to vaccine formulation. Applications of chitosan and its derivatives will be reviewed and their proposed mechanisms in the enhancement of immune responses will be discussed.

Mucosal application of a vaccine can effectively induce both systemic and mucosal immune responses. In general, mucosal applications of antigens result in poor immune responses. Therefore, adjuvant/delivery systems are required to enhance the immune response. Chitosan is a cationic biopolymer which exerts advantages as a vaccine carrier due to its immune stimulating activity and bioadhesive properties that enhance cellular uptake and permeation as well as antigen protection. Similar effects are also shown by chitosan derivatives. In this study, the nanoparticulate systems were prepared by using differently charged chitosan derivatives, N-trimethyl chitosan (TMC, polycationic), and mono-N-carboxymethyl chitosan (MCC, polyampholytic) for mucosal immunisation. The derivatives were synthesised and characterised in-house. The aqueous dispersions of the derivatives were also prepared for comparison. The cytotoxicity studies (MTT assay) on Chinese hamster ovary (CHO-K1) cell lines showed that cell viability was in the order of MCC, chitosan and TMC. Nanoparticles were prepared using ionic gelation method and loaded with tetanus toxoid (TT). Nanoparticles with high loading efficacy (>90% m/m), particle size within the range of 40-400nm, with a negative surface charge for MCC and positive surface charge for TMC and chitosan were obtained. The structural integrity of the TT in the formulations was confirmed by SDS-PAGE electrophoresis analysis. The effective uptake of the FITC-BSA loaded nanoparticles into the cells was demonstrated by cellular uptake studies using J774A.1 cells. Immune responses induced by the formulations loaded with tetanus toxoid were studied in vivo in Balb/c mice. Enhanced immune responses were obtained with intranasal (i.n.) application of nanoparticle formulations. Chitosan and TMC nanoparticles which have positively charged surfaces induced higher serum IgG titres when compared to those prepared with MCC which are negatively charged and smaller in size. Nanoparticle formulations developed in this study can be used as promising adjuvant/delivery systems for mucosal immunisation.

Polymeric scaffold for tissue regeneration was developed for veterinary applications. Oxytetracycline hydrochloride (OTC), which is a widely used antibiotic in veterinary medicine was chosen as the model compound. Gel formulations using chitosan and alginate were prepared in distilled water or in 1% (v/v) acetic acid solution. Sponges were also prepared by a freeze-drying process. Tripolyphosphate was used for cross-linking. Viscosity was decreased in the presence of OTC in chitosan gels whereas no difference was found with alginate gels. All gels showed pseudoplastic behaviour. Water absorption capacity was highest with chitosan/alginate sponges. The solvent used for preparation of the chitosan gels was found to affect the release of OTC. The release of OTC from the sponges was increased by cross-linking. Chitosan/alginate sponges showed the slowest and lowest drug release among the developed sponge formulations in this study. The formulations were found to be biocompatible, inducing no adverse reaction in vivo on surgically formed bone defects of radius of rabbits. The level of organization of the remodelled new bone in the treatment groups was better than that of control. Incorporation of OTC into formulations did not show any considerable enhancing effect.

Local administration of antibiotics in periodontal therapy can be provided with an appropriate delivery system. The purpose of this study was to evaluate the clinical effectiveness of chitosan, both as a carrier in gel form and as an active agent in the treatment of chronic periodontitis (CP). The chitosan gel (1% w/w) incorporated with or without 15% metronidazole was prepared and applied adjunctive to scaling and root planing (SRP) in comparison to SRP alone (control group-C), in CP patients. The clinical parameters such as probing depth (PD), clinical attachment level, the amount of gingival recession, plaque index, gingival index, and gingival bleeding time index were recorded at baseline and at weeks 6, 12, and 24. In all groups, significant improvements were observed in clinical parameters between baseline and week 24 (p < 0.05). The reductions in PD values were 1.21 mm for Ch, 1.48 mm for Ch + M, and 0.94 mm for C groups. No complications related to the chitosan were observed in patients throughout the study period. It is suggested that chitosan itself is effective as well as its combination with metronidazole in CP treatment due to its antimicrobial properties., ((c) 2006 Wiley Periodicals, Inc.)

Oral mucositis is a frequent and potentially severe complication of radiation or chemotherapy for cancer. Associated with atrophy and ulceration of the oral mucosa is an increased risk of infection, and the most common pathogenic agent is Candida. Chitosan is an excellent candidate for the treatment of oral mucositis. Its bioadhesive and antimicrobial properties offer the palliative effects of an occlusive dressing and the potential for delivering drugs, including anti-candidal agents. The aim of this study was to develop an occlusive bioadhesive system for prophylaxis and/or treatment of oral mucositis. Gel and film formulations were prepared using chitosans at different molecular weights and in different solvents. Nystatin, which is considered as a prophylactic agent for oral mucositis was incorporated into the formulations. The in vitro release of nystatin from the formulations was decreased with the increasing molecular weight of chitosan. The effect of the formulations was investigated in vivo in hamsters with chemotherapy-induced mucositis. Mucositis scores in groups treated with nystatin incorporated into gel and suspension formulations were significantly lower (p < 0.05) than those treated with the chitosan gel alone. Survival of animals in the treated groups was higher than that in the control group. The retention time and distribution of the gels in the oral cavity were investigated in healthy volunteers. A faster distribution of nystatin in the oral cavity was obtained using the suspension compared to the gels, but the nystatin saliva level decreased rapidly as well. A drug concentration above the minimum inhibitory concentration (MIC) value for Candida albicans (0.14 microg/ml) was maintained for longer periods of time at the application site (90 min) than at the contralateral site (45 min) in the oral cavity.

Chitosan is a partially deacetylated polymer obtained from the alkaline deacetylation of chitin which is a glucose-based unbranched polysaccharide widely distributed in nature as the principal component of exoskeletons of crustaceans and insects as well as of cell walls of some bacteria and fungi. Chitosan exhibits a variety of physicochemical and biological properties resulting in numerous applications in fields such as waste and water treatment, agriculture, fabric and textiles, cosmetics, nutritional enhancement, and food processing. In addition to its lack of toxicity and allergenicity, and its biocompatibility, biodegradability and bioactivity make it a very attractive substance for diverse applications as a biomaterial in pharmaceutical and medical fields, where it has been used for systemic and local delivery of drugs and vaccines. It also has bioactive properties in its own right. This paper reviews current veterinary applications for chitosan including wound healing, bone regeneration, analgesic and antimicrobial effects. It also discusses the potential application of chitosan to drug and vaccine delivery in veterinary species. Given the restrictions imposed by financial and animal restraint considerations, especially in farming applications, the veterinary drug delivery areas most likely to benefit from chitosan are the delivery of chemotherapeutics such as antibiotics, antiparasitics, anaesthetics, painkillers and growth promotants to mucosal epithelium for absorption for local or systemic activity, and the delivery of immunomodulatory agents to the mucosal associated lymphoid tissue for induction or modulation of local immune responses. The properties of chitosan expected to enhance these functions are discussed, and the future research directions in this field are indicated.

A non-covalently cross-linked palmitoyl glycol chitosan (GCP) hydrogel has been evaluated as an erodible controlled release system for the delivery of hydrophilic macromolecules. Samples of GCP with hydrophobicity decreasing in the order GCP12>GCP11>GCP21 were synthesised and characterised by 1H NMR. Hydrogels were prepared by freeze-drying an aqueous dispersion of the polymer in the presence or absence of either a model macromolecule fluorescein isothiocyanate-dextran (FITC-dextran, MW 4400), and/or amphiphilic derivatives Gelucire 50/13 or vitamin E d-alpha-tocopherol polyethylene glycol succinate. Gels were analysed for aqueous hydration, FITC-dextran release, and bioadhesion, and imaged by scanning electron microscopy. The gels were highly porous and could be hydrated to up to 95x their original weight without an appreciable volume change and most gels eventually eroded. Hydration and erosion were governed by the hydrophobicity of the gel and the presence of the amphiphilic additives. GCP gels could be loaded with up to 27.5% (w/w) of FITC-dextran by freeze-drying a dispersion of GCP in a solution of FITC-dextran. The controlled release of FITC-dextran was governed by the hydrophobicity of the gel following the trend GCP21>GCP11>GCP12. GCP gels were bioadhesive but less so than hydroxypropylmethylcellulose, Carbopol 974NF (7:3) tablets.