Cystatins are able to inhibit the
tumor-associated activity of intracellular
cysteine proteases cathepsins B and L and have been suggested as potential anticancer drugs. We have incorporated
chicken cystatin, a model
protein inhibitor of
cysteine proteases, in
poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) to improve its bioavailability and delivery into
tumor cells.
Cystatin-loaded NPs, 300-350 nm in diameter, were prepared by the double
emulsion solvent diffusion method using low energy emulsification to preserve the
biological activity of the
protein. PLGA NPs and
cystatin-loaded PLGA NPs at concentrations higher than 80 microg/ml were cytotoxic towards MCF-10A neoT cells, but not free
cystatin at concentrations up to 5 microM. To visualize the uptake of
cystatin into living MCF-10A neoT cells, NPs loaded with Alexa Fluor 488-labeled
cystatin were added to the culture medium. They rapidly internalized into the cells, whereas the uptake of free-labeled
cystatin was very slow.
Cystatin, released from the NPs, effectively inhibited
cathepsin B activity, as detected by degradation of specific Z-
Arg-Arg cresyl violet substrate. In contrast, the same amount of free
cystatin showed no inhibition of intracellular
cathepsin B. Our results show that PLGA NPs are a useful carrier system for rapid delivery of
protein inhibitors into
tumor cells, enabling effective inhibition of intracellular proteolysis. The approach can be applied to other
protein drugs active against intracellular targets.