Cellular responses to chitosan in vitro: the importance of deacetylation.

Chitin and chitosan (a deacetylated derivative of chitin) have been proposed for biomedical applications because of their biocompatibility and abundance in nature. We have investigated the effect of the percentage of deacetylation (%DD) of chitosan on biocompatibility from two sources, shrimp and cuttle fish, with two cell lines, L929 and BHK21(C13). The difference in %DD for each source was approximately 10% in the range of 76-90%. Biocompatibility was investigated for: (1) cell adherence and growth on the chitosan samples as substrate; (2) the effect of extract media on 2d and 7d growth; and (3) the presence of an inhibition zone. The results were similar for both cell lines. The chitosan samples were air-dried on to tissue culture-grade petri dishes to provide a substrate for the adherent-cell cultures. The higher %DD substrates from each source supported attachment of the cells, while the lower %DD did not. Cells cultured in medium conditioned by each substrate (i.e. extract medium) displayed an initial difference in growth which was abrogated in cultures incubated for 7 days. No inhibition zone was apparent. However, after 7 days, some cells were noted migrating on to the low %DD substrate disks. The morphology of these cells was changed with the presence of pseudopodia being apparent. Thus, especially with regard to attachment the %DD has a very important effect on the biocompatibility of the chitosan and should be monitored carefully.

Self-assembling of glutathione S-transferase/calmodulin fusion protein on chemically modified gold surface.

The fusion protein technique was used to prepare an artificial polyfunctional protein from calmodulin (CaM) and glutathione S-transferase (GST). The fusion protein was designed, expressed, and then assembled to the glutathione self-assembled gold surface. The protein assembly was confirmed through enzyme binding assay and enzyme immunoassay. Specific binding of the fusion protein to glutathione self-assembled on the gold surface was assessed via a quartz crystal microbalance (QCM). The fusion protein was reversibly adsorbed and desorbed by the competitive binding of glutathione present in a solution, thus showing that the binding of the fusion protein was specific and had a highly oriented molecular configuration.

Calcium responsive two-dimensional molecular assembling of lipid-conjugated calmodulin.

Calmodulin (CaM), a calcium ion sensitive protein, was conjugated with dioctadecyldimethylammonium bromide and subsequently assembled into a monolayer at the air-water interface using the LB method. The lipid-conjugated calmodulin (LCC) retains its calcium sensitivity, determined from the changes in the area-pressure isotherm of the monolayer obtained at the air-water interface. The functionality of this protein assembly was characterized by the activation of phosphodiesterase (PDE), a CaM responsive enzyme. The enzyme activity of PDE coupled with LCC at the air-water interface was measured by using the enzymatic method. It was found that LCC retained its enzyme activity modulating function of calmodulin, which is triggered by calcium ions. This characteristic plays an important role in fabricating molecular assembly of proteins which have a cooperative interaction at the molecular level.