Hyaluronic acid (HA)-tagged pH-sensitive liposomes for intracellular delivery of gemcitabine to pancreatic cancer cells

Abstract

Background and Aim: Pancreatic cancer is one of the most common types of cancer in the world. Gemcitabine (Gem) is the first-line medical chemotherapy for pancreatic cancer. Gem’s resistance is attributable to several processes that occur in pancreatic cancer treatment. The development of multidrug resistance (MDR) has been a major factor responsible for Gem’s chemotherapeutic failure. The ATP-binding cassette (ABC) transporters, such as multidrug resistance protein 5 (MRP5), have been reported to be over-expressed in pancreatic cancer by effluxing Gem out of the cancer cell. To overcome these problems, stable pH-sensitive liposomes (PSL) have been investigated as targeted drug delivery systems. In addition, PSL modified with a ligand hyaluronic acid (HA) for targeting pancreatic cancer cells that have reportedly overexpressed CD44 to increase specific cellular uptake has been investigated. The aim of this study was to investigate and explore the cellular uptake potential of HA-PSL as a drug delivery system for Gem and the effect of curcumin as an MRP5 inhibitor with the potential to overcome drug resistance anticancer properties in pancreatic cancer cell lines. MIA PaCa-2 and a Gem resistant cell line (Gr2000) were used for the cellular uptake study. Methods: A validation and rapid HPLC method was first developed to analyse Gem. Then a Gem derivatization (DNS-Gem) was prepared by using dansyl chloride (DNS-Cl). pHsensitive liposomes (DOPE: DSPC: CHEMS: cholesterol: DSPE-mPEG2000 at a molar ratio of 4:2:2:2:0.3), with and without hyaluronic acid (HA) coating; both were prepared by the thin film-hydration method. HA was then chemically conjugated on the PSL surface. Then the small volume incubation (SVI) method was used for loading the Gem onto both liposomes. To compare the intracellular uptake, the study was carried out using two cell lines, (MIA PaCa-2 and Gr2000) by determining the intercellular concentration, resulting from different liposomes treatment with and without curcumin. Then the drug was extracted by methanol (1:80 v: v) and reaction with DNS-Cl before analysis in HPLC. The ability of HA and the curcumin effect on the intracellular uptake was compared in both cell lines. Results and Discussion: The sensitive HPLC with the special conditions of the derivatization reaction (60℃, 10 min and pH 10.5) makes it a successful method for analysis of DNS-Gem. The intra- and inter-day variations of this method were below 3% with accuracies within 100 ± 1% of the true values. The limit of quantitation (LOQ) and limit of detection (LOD) were 0.37 and 0.12 μM, respectively. The stability of DNS-Gem at room temperature and at 4℃ was observed. Both were found to be stable. pH-sensitive liposomes with and without HA coating were prepared and then the Gem was loaded in both liposomes by the SVI method. The characteristics of both liposomes (HA-PSL and non-HA-PSL) were studied; the results showed that the zeta potential was higher in the HA-PSL than in the non-HA-PSL. But the particle sizes of both liposomes were around 150nm. The results also showed that both liposomes have the same value of EE (26.3 ± 0.8% for HA-PSL, and 26.6 ± 2.4% for non-HA-PSL (n = 3)) with similar value of DL (3.9 ± 0.1% for HA-PSL, and 4.0 ± 0.3%, (w: w) for non-HA-PSL). The release profiles were higher in the non-HA-PSL irrespective of having the same drug levels in the liposomes. The cellular uptake study of Gem was investigated. It was found that the six different formulations were significantly different in both MIA PaCa-2 and Gr2000. From the results, the co-treatment HA-PSL with curcumin had a higher drug delivery of Gem than the formulation type without curcumin (HA-PSL, non-HA-PSL and Gem solution). The results also show that the uptake of the Gem concentration was higher in MIA PaCa-2 pancreatic than in the Gr2000 pancreatic cell lines. This provided the rationale that the combination of HAPSL and curcumin appeared to be most effective in increasing the cellular uptake of Gem by MIA PaCa -2 cells, and in particular the resistance cells. Conclusion: The HA-PSL improved the selective intercellular delivery of Gem, which led to a significantly greater Gem accumulation within the cancer cell. In addition, curcumin showed the potential to further improve Gem uptake by pancreatic cancer. Thus, HA-tagged PSL combined with curcumin exhibited a potent strategy to overcome the Gem-resistance by enhancing the intracellular delivery of Gem to pancreatic cancer cells. Future animal study is planned to investigate the in vivo targeting property and anticancer efficacy.