Potential Combination Therapies for Gemcitabine-resistant Pancreatic Cancer Facilitated with Intracellular Delivery with Smart Liposomes: Formulation and In Vitro Evaluation

Abstract

Background: Pancreatic cancer, most likely presented as pancreatic ductal adenocarcinoma (PDAC), is the most lethal human malignancy. Gemcitabine has been the first-line chemotherapy drug for patients with locally advanced or metastatic PDAC. However, the conventional gemcitabine formulation either alone or in combination with other drugs encounters many barriers primarily drug resistance and side effects to patients. In the past decade, tumour targeted drug delivery with nanotechnology has emerged to improve the chemotherapeutic efficacy and liposomes are regarded as the optimal carriers. Among various liposomes, the pH-sensitive liposomes (pSL) are designed to be a good candidate for tumour targeted delivery, with the ability to be stable at physiological pH (blood circulation and normal tissue), but undergo destabilisation under acidic conditions (tumour tissues). Furthermore, liposomes can be used to increase drug solubility. Aim: This study aimed to develop new pSL formulations containing bufalin, a steroid with anti-tumour activity on PDAC, or 4-methylumbelliferone (4-MU), an inhibitor for hyaluronic acid (HA), and investigate their potential to overcome gemcitabine resistance in pancreatic cancer. It was hypothesised that bufalin would be a possible candidate to overcome gemcitabine resistance due to its multiple anti-tumour effects and its function on pancreatic cancer stem cells. It was also hypothesised that 4-MU might overcome gemcitabine resistance by inhibiting HA synthesis or enhancing the effects of gemcitabine. Methods: The reverse phase HPLC methods were firstly developed and validated for developing the following formulations. For both bufalin and 4-MU formulations, pSL formulations were comprised of DOPE: DSPC:(cholesterol): CHEMS: DSPE-PEG2000 with a molar ratio of 4:2:(2):2:0.3 in 10 mg/ml lipids. Both bufalin and 4-MU were passively loaded into the liposomal bilayer through the thin film hydration method, while gemcitabine was loaded into the liposomal core by the small volume incubation method. The physicochemical properties of two formulations were evaluated over the Malvern Zetasizer, the drug loading (DL)) and entrapment efficiency (EE) were determined and calculated by the HPLC analysis. The in vitro pH-responsive release study of two formulations was based on the dialysis method. The in vitro cytotoxicity study was carried out by the methyl-thiazolyl-diphenyl-tetrazolium bromide (MTT) or 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assay on the gemcitabine resistant pancreatic adenocarcinoma cell line (Gr2000) and its parental MIA PACA-2 cell line as control. Results: HPLC methods for analysing bufalin and 4-MU were linear and validated, suitable for determination during formulation. The resulted particle size, PDI and zeta potential for bufalin-pSL was 110.9 nm, 0.109 and -15.0 mV respectively; while those results for GEM-4-MU-pSL were respectively 150.1 nm, 0.055 and -15.0 mV. Both formulations were stable for 2 months at 4 °C in dark except for a significant decrease in zeta potential for GEM-4-MU-pSL. Moreover, both formulations demonstrated slower release rates at pH 7.4 than pH 5.0, indicating pH responsiveness. The in vitro cytotoxicity study revealed that bufalin-pSL was potent to Gr2000 cell line, with an IC50 of 1.1 nM and could eliminate all Gr2000 cell at concentration of 100 nM; Moreover, its cytotoxicity effect could be further enhanced when combined with GEM-pSL with IC50 of 0.4 nM. On the other hand, 4-MU also exhibited cytotoxicity effects on both MIA PACA-2 and Gr2000 cells and enhanced the cytotoxicity of GEM-pSL on Gr2000 cell line when combined in GEM-4-MU-pSL, as seen a decreased IC50 of gemcitabine by 2 fold, (1125 nM vs 2077 nM of GEM-pSL), and a smaller group of cells survived (3% vs 5%) under the same maximal concentration of gemcitabine. Conclusion: This thesis demonstrated the great potential of the combination of bufalin or 4-MU with gemcitabine to treat gemcitabine-resistant PC, by targeting the different signalling pathways. The use of pSL enabled the poorly soluble drugs to be formulated into an injectable form, and targeted delivered into the cancer cells, thus increase the efficacy.