Abstract:
Background: Pancreatic cancer (PC) is one of the most aggressive tumours and gemcitabine is the first-line medical chemotherapy. However, conventional formulations suffer from low tumour-specificity, often resulting in severe side effects. Furthermore, the development of multidrug resistance (MDR) has been a major factor responsible for gemcitabine’s chemotherapeutic failure. The ATP-binding cassette (ABC) transporters, such as multidrug resistance protein 5 (MRP5), have been reported to be over-expressed in PC and may facilitate the MDR by effluxing gemcitabine out of the cancer cell. Liposomes, particularly the PEGylated, have been investigated as effective drug carriers to confer better tumour targeting by exploiting the enhanced permeability and retention (EPR) effect. Liposomes with a large payload may enter cancer cells via endocytosis and thus may bypass the efflux pumps. Furthermore, pH-sensitive liposomes (PSL) may further enhance the cytoplasmic drug delivery, enhancing its availability to its DNA target. The ultimate aim of this project was to investigate a newly designed multi-functional PSL to circumvent gemcitabine resistance in PC: the long-circulation and pH-sensitivity would be used for tumour targeting followed by cytoplasmic drug delivery to cancer cell; while the codelivery of a MRP5 inhibitor, curcumin would overcome the drug efflux effect. Methods: A rapid and reliable gradient high performance liquid chromatography (HPLC) method was developed for the simultaneous analysis of the hydrophilic gemcitabine and the lipophilic curcumin with the aid of the Central Composite Design (CCD). Stable non-pH sensitive conventional liposomes (NPSL) and PSL, both coated with polyethylene glycol on the surface (PEGylation), were prepared by the thin film hydration and extrusion method. To increase the drug entrapment efficiency (EE) and drug loading (DL), a novel passive drug loading approach, Small Volume Incubation (SVI) method, was developed and compared to the remote loading method using a transmembrane ammonium sulphate gradient. To retain the pH-sensitivity that was compromised by PEGylation, a post-insertion technique was compared with pre-insertion. For the dual-drug delivery systems, curcumin was loaded into liposome bilayers via passive loading while gemcitabine was loaded in the aqueous cores using a modified SVI method. Confocal laser scanning microscopy coupled with a calcein self-quench assay was applied, for the first time, to evaluate the endosome escape capability of the liposomes. To predict the potential for long-circulation in vivo resulting from different degrees of PEGylation, an intracellular uptake study was carried out using macrophages. The ability of curcumin to reverse gemcitabine resistance was evaluated by measuring its effects on gemcitabine intracellular accumulation and cytotoxicity to gemcitabine-resistant PC cell lines (MIA PaCa-2 and PANC-1). Similarly, formulation factors, such as DL, methods for PEGylation and the effect of curcumin were all compared with regard to their cytotoxicities in these drug-resistant PC cells and their pharmacokinetics in rats compared with drug solutions. Results and Discussion: An HPLC method for the simultaneous analysis of hydrophilic gemcitabine and lipophilic curcumin was established with the aid of CCD, which resulted in sharp peaks and retention times < 11 min. The intra- and inter-day variations were ≤ 5%, with accuracies between 90 - 110% of the true values. The limit of quantitation (LOQ) and limit of detection (LOD) for both compounds were 0.03 and 0.01 μM, respectively. Using the optimized SVI method, an increased EE (37 ± 1%) and DL (4.2 %, w/w) were obtained for NPSL with a particle size of 200 ± 5 nm and zeta potential of -15 ± 0.3 mV. In contrast, the EE for remote loading was 12.3 ± 0.3% and a burst release was observed possibly due to the higher intra-liposomal osmotic pressure created by ammonium sulphate (550 mOsm). Both PEGylation (0.5-5% mol) and incorporation of curcumin into the liposomal bilayers reduced the pH-sensitivity in a concentration-dependent manner. This is attributable to two possible factors: i) the increased viscosity of the intra-liposome cores and the reduced fluidity of the liposome bilayer which may inhibit the transformation of the pH-sensitive components; ii) the reduced zeta potential resulted in less attraction with H+ for transformation of membrane. At equal concentrations, the cytotoxicity of gemcitabine alone formulations towards MIA PaCa-2 cells was ranked in the order: PSL (DL 0.5%) = PSL (DL 4.5%) > NPSL (DL 4.2%) > NPSL (DL 0.5%) ≥ gemcitabine solution. The enhanced cytotoxicity of PSL was attributed to more effective cytoplasmic drug release via endosome escape. The NPSL with a gemcitabine DL of 0.5% did not show any benefits over gemcitabine solution, suggesting a high DL is essential to overcome the subsequent lysosomal degradation. The macrophage uptake study showed that PEGylation (1-5%) reduced the uptake of PSL by macrophages by over 60%, suggesting the long-circulation property as a result of PEGylation. PEGylation, particularly with the traditional pre-insertion technique also reduced the pH-sensitivity which was enhanced by the post-insertion technique. This resulted in PEGylation only on the outer surface which may facilitate the transformation of pH-sensitive components at acidic conditions. The cytotoxicity to MIA PaCa-2 cells was ranked in the order: post-inserted PSL ≥ pre-inserted PSL > gemcitabine solution, which was consistent with the confocal microscopic observation of intra-cytoplasmic delivery of calcein. Curcumin encapsulation had no effect on the liposome size. PSL loaded with gemcitabine with or without curcumin exhibited no change in size during 3 months storage at 4 oC in either suspension or pellet form, but an increased zeta potential from -15 to -30 mV was observed after two months. The chemical stability of gemcitabine was retained in both storage forms; whereas curcumin was significantly (p < 0.05) degraded to 71.2% after three months in suspension due to leakage and subsequential degradation, but no significant change was observed if stored as a pellet. The cytotoxicity of the gemcitabine-curcumin loaded PSL towards MIA PaCa-2 cells was significantly higher than any of the single drug loaded PSL or solutions, which was consistent with the curcumin effect on gemcitabine cellular accumulation. The in vitro data suggested that MRP5 might play a role in gemcitabine's resistance in PC and that curcumin had the ability to reverse such resistance. In rats, both pre- and post-inserted PEGylated PSL resulted in significant reductions (p < 0.05) in gemcitabine’s plasma clearance (58.6 and 38.4 ml/h/kg) and significant increases in the AUC (56.9 and 87.1 μM·h) and half-life (6.1 and 6.2 h) compared to gemcitabine solution (152.9 ml/h/kg, 22.2 μM·h and 1.4 h). The presence of curcumin in the dual-loaded PSL did not influence gemcitabine’s pharmacokinetics in rats. The PSL significantly reduced curcumin’s plasma clearance (3357 ml/h/kg) and volume distribution (13852 ml/kg), resulting in an approximate 4-fold increase in the AUC (1.5 μM·h) in contrast to its solution (16280 ml/h/kg, 54561 ml/kg and 0.4 μM·h). Conclusion: The multi-functional gemcitabine-curcumin loaded PSL exhibited the most effective capability to reverse gemcitabine-resistant in PC cells by enhancing intracytoplasmic drug delivery. The nano-liposomal system also improved the pharmacokinetics of both encapsulated compounds, likely leading to better tumour targeting via the EPR effect, greater gemcitabine accumulation within the cancer cell, and thus has the potential to improve efficacy for this front-line drug in PC treatment. Technically, remote loading using ammonium sulphate gradient is not suitable for gemcitabine. Additional requirements to the current criteria for remote loading using ammonium sulphate gradient (pKa < 11) for basic drugs with a pKa > 4.6 and intra-liposomal precipitation were suggested. In addition, PEGylation by post-insertion offered advantages over pre-insertion to obtain PSL with enhanced pH-sensitivity, more effective intra-cytoplasmic delivery, and superior pharmacokinetics.