Insulin, glucose oxidase and positively charged star polymers were incorporated into multilayer films by the layer-by-layer (LbL) assembly method. It is interesting to find that the arrangement sequence of the three components could significantly affect the glucose-responsive controlled release behaviors. The insulin release in vitro could be tuned to linear release and obtain desired "on-off" sensitivity in response to stepwise glucose challenge, just by rearranging the assembly sequence of LbL building blocks. Further, the controlled release of insulin in vivo, as well as the hypoglycemic effect, could be obviously prolonged from 17days to 36days by this simple strategy without changing the dosage of all the LbL components. In addition to provide a potential glucose-responsive delivery system for insulin, the strategy described in this paper could be valuable for various drug-incorporated LbL systems with three or more components.
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