02 BOOK OF ABSTRACTS .indd

Nanoscale carriers facilitate delivery of protein drugs into
tumour cells
Mateja Cegnar, Julijana Kristl and Janko Kos
Faculty of Pharmacy, University of Ljubljana, A{ker~eva 7, Ljubljana, Slovenia
Advances in molecular biology, biotechnology and medicinal chemistry have made
available a number of new anticancer drugs, however, it is clearly recognized that
inadequate delivery is the single most important factor delaying their application in
clinical practice.
In our study we developed a nanoscale polymer carrier, in order to increase the
bioavailability of protein drug as the carrier protects it from rapid degradation in
biological environment and facilitates its intercellular delivery. Cystatin was selected
as a model protein drug due to its high potential for inactivating cysteine proteases,
involved in processes of tumour invasion and metastasis. As reported, extracellular
and intracellular cysteine proteases participate in these processes, therefore the
inhibition of both fractions would have high potential to impair malignant process.
A biodegradable copolymer of lactic and glycolic acid (PLGA) was used as a
carrier material, which was transformed into nanoparticles (NPs) by using lowenergy
double emulsion solvent diffusion method. NPs had size of 300-350 nm
diameter, and contained 1.6% (w/w) of cystatin, retaining 85% of its starting activity.
To follow cellular uptake of nanoparticles, cystatin was labelled with fluorescent
dye (Alexa Fluor 488) prior to its encapsulation into NPs. Image analysis showed
rapid internalization of NPs into MCF-10A neoT cells as the fluorescence spots were
detected immediately after treatment with NPs. On the other hand, labelled free
cystatin was internalised very slowly, suggesting that NPs facilitate the delivery of
its cargo into the cell.
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To examine the capability of nanoparticle-delivered cystatin to inhibit its lysosomal
targets a fluorogenic substrate Z-Arg-Arg Cresy Violet was used. The substrate
easily penetrates cell membranes and is degraded into red fluorescent product after
cleavage with cathepsin B. Living cells developed strong red fluorescence in the
perinuclear region after treatment with the substrate. In case when cells were pretreated
with cystatin-loaded NPs, no red fluorescence developed, showing effective
intracellular inhibition. Again, free cystatin did not inhibit cathepsin B activity as did
not empty NPs.
Our results show that PLGA NPs represent a useful carrier system for rapid
delivery of protein inhibitors into cells, enabling effective inhibition of intracellular
proteolysis.