The effect of cross-linking of chitosan microspheres with genipin on protein release

Genipin, a natural and non-toxic cross-linking reagent, was evaluated for its effects on the drug/protein release and swelling of chitosan microspheres. Chitosan microspheres, using albumin as a model protein, were prepared and cross-linked with 0.5 mM genipin for 4 to 16 h or for 4 h using 0.5 to 2.0 mM genipin. The degree of cross-linking, swelling and the release of albumin from the microspheres was determined by the ninhydrin assay, measuring change in mass between dry and wet spheres, and in 31-day elution tests, respectively. The degree of cross-linking increased up to maximum of 33% to 34% with up to 8 hour cross-linking time or with up to 1.0 mM genipin concentration. Additional cross-linking time or concentration did not significantly increase degree of cross-linking. Swelling ratios decreased significantly from 119.2% in the uncross-linked condition to 108.8% at 16 h cross-linking time. However, increasing the genipin concentration resulted in much smaller decreases in swelling. The release of albumin was reduced with as little as 4 h cross-linking time to 30.9% of uncross-linked microspheres for up to 24 days and by as much as 52.3–60.0% for up to 31 days with 8–16 h cross-linking time. Using genipin concentrations of 1.0 to 2.0 mM for 4 h, greatly reduced albumin release to only 12.4% to 27.1% on day 24. These data demonstrate that protein and drug delivery rates from chitosan microspheres may be controlled and extended by controlling the degree of cross-linking with genipin.