This may be because cytochrome c is larger than insulin. From the surface area of the HA (>9.4m2/g) and the protein diameters (d) of insulin (3nm) and cytochrome c (4nm) [12], the percent of occupied area can be estimated. In the experiment, 0.5mg of proteins, that is 9 × 10-8mol (5 × 1016 molecules) of insulin and 4 × 10-8mol (2 × 1016 molecules) of cytochrome c, were used. The projected area of proteins may be approximated as π(d/2)2, assuming the protein Inhibitors,research,lifescience,medical to be spherical. Given that all molecules were absorbed, the occupied areas of insulin and cytochrome c were 0.4m2 and 0.3m2, respectively. The surface area of HA (10mg, 20mg,

and 30mg) can be JNK-IN-8 nmr calculated as >0.094m2, >0.19m2, and >0.28m2, respectively. It is suggested that the surface area of HA was fully occupied by proteins. Inhibitors,research,lifescience,medical Even though the absorption amount of

cytochrome c could be correlated to the total surface area of HA, that of insulin could not. It is possible that insulin was absorbed to form multilayered structures. Figure 1 HPLC analysis. (a) Chromatograms of cytochrome c (top) and insulin (bottom). (b) Correlations between the peak area and the concentrations of cytochrome c (solid symbols) and insulin (open symbols). Figure 2 Association of cytochrome c (solid symbols) and insulin (open symbols) with various amounts of HA after 4h. Figure 3 indicates time-dependent association of cytochrome Inhibitors,research,lifescience,medical c to HA. Long-term incubation contributed to the efficient loading of cytochrome c even with a small amount of HA. Figure 3 also indicates that cytochrome c bound to HA in two Inhibitors,research,lifescience,medical phases. The initial absorption

occurred in less than 1h, and the subsequent absorption occurred more slowly, in the range of an hour. The first absorption phase might be attributed to surface absorption and the latter by penetration into the pores. The release of cytochrome c was also examined at different incubation times. The release profiles also occurred over two phases, in less than 1h and over the hour Inhibitors,research,lifescience,medical range (Figure 3), similar to the adsorption profile. This result suggests that absorbed proteins at 4��8C the surface were released very fast and those within the pores were released more slowly. Thus, regulation of release can be achieved by the control of protein size and the pore size of HA. It is possible that step-by-step protein release can be performed in an HA-based delivery system. Figure 3 Time-dependent association (solid symbols) and dissociation (open symbols) of cytochrome c with HA (20mg). Figure 2 shows that insulin was readily bound to HA. We also investigated the release profiles of insulin (Figure 4). Less than 40% of the bound insulin was released from HA, even though 70% of cytochrome c was released after 24h. Insulin is smaller than cytochrome c and readily bound to HA (Table 1 and Figure 2), but the release of insulin is slower than that of cytochrome c (Figure 4).