Can intracellular drug delivery using hyaluronic acid functionalised pH-sensitive liposomes overcome gemcitabine resistance in pancreatic cancer?

Chemoresistance poses a major challenge in cancer treatment. This study aims to investigate whether intracellular drug delivery using hyaluronic acid (HA) functionalised pH-sensitive liposomes (HA-pSL) can circumvent gemcitabine resistance in pancreatic cancer (PC). HA-pSL were obtained by covalently conjugating HA with preformed pSL. A resistant PC cell line Gr2000 was developed by exposing MIA PaCa-2 cells to gemcitabine, and characterised for their expression of CD44, a receptor for HA, and drug transporters. Cellular uptake and intracellular trafficking of liposomes were determined by confocal microscopy and HPLC analysis of intracellular drug content. Following a pharmacokinetic study in rats, anti-tumour efficacy was compared between MIA PaCa-2 and Gr2000 xenograft mouse models. HA-pSL with an HA density of 179 μg/μmol had a larger size (152.3 vs 136.3 nm), and higher zeta potential (-46.8 vs -10.5 mV) than pSL. The sensitivity of Gr2000 to gemcitabine reduced 444 times compared to its parental cell line, despite no change to the total drug influx, as drug influx- and efflux-transporters in Gr2000 cells were simultaneously up-regulated. Both cell lines had high expression of CD44. HA facilitated cell uptake without compromising the endosome-escape ability of pSL as evidenced by confocal images and co-localization analysis of the dual-fluorescence labelled liposomes and Lysotracker. HA-pSL significantly outperformed pSL, and increased cellular drug influx by 3.6 times in MIA PaCa-2 cells, and 4.6 times in Gr2000 cells. Both liposomes improved the pharmacokinetic profile of free drug. HA-pSL treatment was superior to pSL, and resulted in 6.4 times smaller tumours (weight) in the MIA PaCa-2 xenograft models, and 3.1 smaller in the Gr2000 models compared with the free drug. Taken together, this study highlighted the use of intracellular delivery strategies (HA-CD44 interaction and endosome escape) to overcome gemcitabine resistance, however, the overall improvement was marginal and tumours still existed. Further improvement in delivery efficiency of HA-pSL to target tumours and additional manipulation of the cellular metabolism of gemcitabine are needed to tackle chemoresistance.