Chitosan as a growth stimulator in orchid tissue culture

The effect of shrimp and fungal chitosan on the growth and development of orchid plant meristemic tissue in culture was investigated in liquid and on solid medium. The growth of meristem explants into protocorm-like bodies in liquid medium was accelerated up to 15 times in the presence of chitosan oligomer, the optimal concentration being 15 ppm. The 1 kDa shrimp oligomer was slightly more effective compared to 10 kDa shrimp chitosan and four times more active compared to high molecular weight 100 kDa shrimp chitosan. The 10 kDa fungal chitosan was more effective compared with 1 kDa oligomer. The development of orchid protocorm into differentiated orchid tissue with primary shoots and roots was studied on solid agar medium. The optimal effect, the generation of 5–7 plantlets in 12 weeks was observed in the presence of 20 ppm using either 10 kDa fungal or 1 kDa oligomer shrimp chitosan. The data are consistent with preliminary results from field experiments and confirm unequivocally that a minor amount of chitosan has a profound effect on the growth and development of orchid plant tissue.     

Film-forming process and biocide assessment of high-molecular-weight chitosan as determined by combined ATR-FTIR spectroscopy and antimicrobial assays

This pioneering study reported about the film-forming properties of high-molecular-weight chitosan as followed in situ by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, and has implications in fields such as biomedical, pharmaceutical, packaging, and coating applications. From the results, it was observed that immediately after dissolution in an acetic acid aqueous solution and subsequent casting over the ATR crystal, the formed carboxylate antimicrobial (-NH3+ -OOCH) species are not stable in the film formulation and become reduced over time; further assays confirmed previous research, which suggested that the presence and stability of these groups is strongly dependent, among other factors, on storage conditions. As-received chitosan and chitosan neutralized in NaOH films did not exhibit biocide performance towards Staphylococcus aureus. The antimicrobial tests were also found to strongly relate the presence of a sufficient quantity of these carboxylate groups to the chitosan activity as a biocide agent. Moreover, a novel methodology based on the use of a normalized infrared band centered at 1405 cm(-1) is proposed which can be correlated with the antimicrobial character of the biopolymer.     

Alginate-chitosan complex coacervation for cell encapsulation: effect on mechanical properties and on long-term viability

The use of chitosan in complexation with alginate appears to be a promising strategy for cell microencapsulation, due to the biocompatibility of both polymers and the high mechanical properties attributed by the use of chitosan. The present work focuses on the optimization and characterization of the alginate-chitosan system to achieve long-term cell encapsulation. Microcapsules were prepared from four types of chitosan using one- and two-stage encapsulation procedures. The effect of reaction time and pH on long-term cell viability and mechanical properties of the microcapsules was evaluated. Using the single-stage encapsulation procedure led to increase of at least fourfold in viability compared with the two-stage procedure. Among the four types of chitosan, the use of high molecular weight (MW) chitosan glutamate and low MW chitosan chloride provided high viability levels as well as good mechanical properties, i.e., more than 93% intact capsules. The high viability levels were found to be independent of the reaction conditions when using high MW chitosan. However, when using low MW chitosan, better viability levels (195%) were obtained when using a pH of 6 and a reaction time of 30 min. An alginate-chitosan cell encapsulation system was devised to achieve high cell viability levels as well as to improve mechanical properties, thus holding great potential for future clinical application.     

Determination of binding constants of lipopolysaccharides of different structure with chitosan

The interaction of endotoxins--lipopolysaccharides (LPS) different in degree of the O-specific chain polymerization--with 20- and 130-kD chitosan was studied using the competitive binding of LPS with the complex of chitosan-anionic dye (tropaeolin 000-2) and the direct binding of (125)I-labeled LPS with chitosan immobilized on Sepharose 4B. The interaction of 20-kD chitosan with LPS was non-cooperative, and immobilization of the polycation on Sepharose resulted in its binding to (125)I-labeled LPS with a positive cooperativity. The interaction of LPS possessing a long O-specific chain with 130-kD chitosan was characterized by negative cooperativity. Binding constants of LPS with the polycation and the number of binding sites per amino group of chitosan were determined. The interaction affinity and stoichiometry of the LPS-chitosan complexes significantly depend on the LPS structure and concentration in the reaction mixture. The increase in the length of carbohydrate chains of LPS results in increase in the binding constants and decrease in the bound endotoxin amount.     

Oral delivery of curcumin bound to chitosan nanoparticles cured Plasmodium yoelii infected mice

Curcumin has been shown to have anti malarial activity, but poor bioavailability and chemical instability has hindered its development as a drug. We have bound curcumin to chitosan nanoparticles to improve its bioavailability and chemical stability. We found that curcumin bound to chitosan nanoparticles did not degrade that rapidly in comparison to free curcumin when such particles were incubated in mouse plasma in vitro at room temperature. The uptake of bound curcumin from chitosan nanoparticles by mouse RBC was much better than from free curcumin. Oral delivery of curcumin bound chitosan nanoparticles to normal mice showed that they can cross the mucosal barrier intact and confocal microscopy detected the nanoparticles in the blood. Curcumin loaded chitosan nanoparticles when delivered orally improved the bioavailability of curcumin in the plasma and RBC. While mice infected with a lethal strain of Plasmodium yoelii (N-67) died between 8 and 9 days post infection, feeding of chitosan nanoparticles alone made them to survive for five more days. Feeding 1mg of native curcumin to infected mice per day for seven days resulted in survival of one third of mice but under the same condition when 1mg of curcumin bound to chitosan nanoparticles was fed all the mice survived. Like chloroquine, curcumin inhibited parasite lysate induced heme polymerization in vitro in a dose dependent manner and curcumin had a lower IC(50) value than chloroquine. We believe that binding of curcumin to chitosan nanoparticles increases its chemical stability and enhances its bioavailability when fed to mice. In vitro data suggest that it can inhibit hemozoin synthesis which is lethal for the parasite.     

Synthesis and characterization of chitosan-homocysteine thiolactone as a mucoadhesive polymer

Two mucoadhesive thiolated polymers were synthesized by the covalent attachment of homocysteine thiolactone (HT) to chitosan and N,N,N-trimethyl-chitosan (TM-chitosan) at various chitosan:HT ratios. The amount of thiol and disulphide groups immobilized on the chitosan influenced the polymer's mucoadhesion positively and negatively, respectively, with the optimal chitosan:HT (w/w) ratio being found to be 1:0.1. The interaction between mucin and chitosan and its three derivatives was highest for the thiolated chitosan derivatives but was pH dependent. HT-chitosan and TM-HT-chitosan, with the thiol groups of 64.15 and 32.48 μmol/g, respectively, displayed a 3.67- and 6.33-fold stronger mucoadhesive property compared to that of the unmodified chitosan at pH 1.2, but these differences were only ∼1.7-fold at pH 6.4. The swelling properties of TM-HT-chitosan and HT-chitosan were higher than that of chitosan and TM-chitosan, attaining a swelling ratio of up to 240% and 140%, respectively, at pH 1.2 within 2 h.     

Chitosan-based heterogeneous catalysts for enantioselective Michael reaction

Novel chitosan-supported cinchona alkaloids have been developed as heterogeneous catalysts for enantioselective Michael reaction. As-synthesized products as organocatalysts for asymmetric Michael reaction have a high efficiency, providing highly functionalized products (containing adjacent quaternary and tertiary stereocenters) with good stereoselectivity (up to 93% enantiomeric excess) in high yields and recyclability (up to five runs).     

Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

Photochemical tissue bonding (PTB) is a sutureless technique for tissue repair, which is achieved by applying a solution of rose bengal (RB) between two tissue edges^1,2. These are then irradiated by a laser that is selectively absorbed by the RB. The resulting photochemical reactions supposedly crosslink the collagen fibers in the tissue with minimal heat production³. In this report, RB has been incorporated in thin chitosan films to fabricate a novel tissue adhesive that is laser-activated. Adhesive films, based on chitosan and containing ~0.1 wt% RB, are fabricated and bonded to calf intestine and rat tibial nerves by a solid state laser (λ=532 nm, Fluence~110 J/cm², spot size~0.5 cm). A single-column tensiometer, interfaced with a personal computer, is used to test the bonding strength. The RB-chitosan adhesive bonds firmly to the intestine with a strength of 15 ± 6 kPa, (n=30). The adhesion strength drops to 2 ± 2 kPa (n=30) when the laser is not applied to the adhesive. The anastomosis of tibial nerves can be also completed without the use of sutures. A novel chitosan adhesive has been fabricated that bonds photochemically to tissue and does not require sutures.     

Dynamics of diffusivity and pressure drop in flow-through and parallel-flow bioreactors during tissue regeneration

In this study, transport characteristics in flow-through and parallel-flow bioreactors used in tissue engineering were simulated using computational fluid dynamics. To study nutrient distribution and consumption by smooth muscle cells colonizing the 100 mm diameter and 2-mm thick scaffold, effective diffusivity of glucose was experimentally determined using a two-chambered setup. Three different concentrations of chitosan-gelatin scaffolds were prepared by freezing at -80°C followed by lyophilization. Experiments were performed in both bioreactors to measure pressure drop at different flow rates. At low flow rates, experimental results were in agreement with the simulation results for both bioreactors. However, increase in flow rate beyond 5 mL/min in flow-through bioreactor showed channeling at the circumference resulting in lower pressure drop relative to simulation results. The Peclet number inside the scaffold indicated nutrient distribution within the flow-through bioreactor to be convection-dependent, whereas the parallel-flow bioreactor was diffusion-dependent. Three alternative design modifications to the parallel-flow were made by (i) introducing an additional inlet and an outlet, (ii) changing channel position, and (iii) changing the hold-up volume. Simulation studies were performed to assess the effect of scaffold thickness, cell densities, and permeability. These new designs improved nutrient distribution for 2 mm scaffolds; however, parallel-flow configuration was found to be unsuitable for scaffolds more than 4-mm thick, especially at low porosities as tissues regenerate. Furthermore, operable flow rate in flow-through bioreactors is constrained by the mechanical strength of the scaffold. In summary, this study showed limitations and differences between flow-through and parallel-flow bioreactors used in tissue engineering.