Investigation of Hyaluronic Acid-Functionalised pH-Sensitive Liposomes to Overcome Gemcitabine Resistance in Pancreatic Cancer: Mono- and Dual-Therapy

Abstract

Background: Pancreatic cancer (PC) has the highest rate of mortality among all cancers. Gemcitabine is a first line chemotherapeutic agent for PC, however, the lack of tumour specificity and chemo-resistance pose major challenges to PC treatment. The lack of gemcitabine influx transporters and overexpression of gemcitabine efflux transporters have been found to confer resistance to gemcitabine in PC. In addition, alternations of gemcitabine metabolism enzymes have also been associated with drug resistance in PC. In the field of drug delivery, nanoparticles have been investigated for tumour targeted drug delivery based on the so-called enhanced permeability and retention (EPR) effect. Among these nanoscaled systems, pH-sensitive liposomes (pSL) have demonstrated the ability to release their payload inside tumour cells as they destabilise at a low pH such as in the endosomes. Furthermore, surface modification of liposomes with a ligand, for instance hyaluronic acid (HA), to target receptors (e.g. CD44) overexpressed on cancer cells, has been used to achieve active tumour targeting via endocytosis, resulting in improved drug delivery to cancer cells. Aim: This thesis aimed to investigate whether a dual intracellular delivery strategy using pSL grafted with HA (HA-pSL) could circumvent gemcitabine resistance in PC. Secondly, we investigated whether co-delivery of gemcitabine with an inhibitor of multidrug resistanceassociated protein 5 (MRP5) or cytidine deaminase (CDA) using HA-pSL would further increase the potential for overcoming gemcitabine resistance. It was hypothesised that the CD44-HA interactions would promote internalisation of liposomes into PC cells; upon entering the cells, pH-sensitivity will allow prompt release of gemcitabine from late endosomes (pH 5.0-5.5) (endosomal escape) to the DNA target of drug at sufficient levels to kill the resistant cells. In addition, An agent directly working on gemcitabine efflux transporters or favourably interfering drug deactivation was co-delivered, thus maintaining cellular drug concentration for higher potency. Results and discussion: A resistant cell line Gr2000 was successfully developed with 444 times reduced sensitivity to gemcitabine compared to MIA PaCa-2 but similar morphology and growth rate to MIA PaCa-2. Furthermore, after 72 h exposure to high gemcitabine concentration, 20% cells remained alive in the Gr2000, compared to 5 % in MIA PaCa-2, possibly revealing a gemcitabine resistance population (RP) with cancer stem cell properties. MTT cell viability assay with a seeding density of 800 cells/well was chosen to determine cytotoxicity in the formulation development in this thesis. Importantly, both MIA PaCa-2 and Gr2000 cell lines showed high expression of CD44, verifying the use of HA on liposomes for promoting intracellular drug delivery in this PhD study. Following chemical conjugation of plain pSL with HA solution at 4 mg/mL, the HA coating efficiency of HA-pSL was 43.2 ± 1.6% (n=3), while the HA density was 178.9 μg/μmol (HA to total lipids), resulting in significantly increased cell uptake (following 3 h exposure) by MIA PaCa-2 cells compared to pSL. Further increase in HA concentration up to 10 mg/mL did not further enhance the cellular uptake. The final 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 plain pSL. HA facilitated cellular uptake reaching the maximal level at 1h, without compromising the endosome escape ability of pSL as evidenced by confocal images and co-localisation 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 in tumour growth inhibition, and resulted in 6.4 times smaller tumours (weight) in the MIA PaCa-2 xenograft models, but only 3.1 smaller in the Gr2000 models compared with the free drug treatment. To further develop strategies to improve efficacy in resistant PC, biological resistance mechanisms in Gr2000 were further investigated. RT-PCR analysis revealed that mRNA expression of drug influx transporters (hENT1, hCNT1) and efflux transporters (MRP5) in Gr2000 cells were simultaneously up-regulated. Expression of CDA, RRM1 and RRM2 was up-regulated while that of dCK was down-regulated in Gr2000. Also, protein expression of dCK was found to be hardly detectable in Gr2000 in contrast to the high level in MIA PaCa-2. Therefore, in this thesis, consequently co-delivery of gemcitabine and an agent that inhibited MRP5 efflux transporters or CDA enzyme into the HA-pSL were further employed to overcome chemo-resistance. An optimised DL of curcumin, 0.96 % (w/w) was achieved for the gemcitabine and curcumin co-loaded HA-pSL, which had increased size of 33 nm but similar zeta-potential. The dual-drug delivery HA-pSL showed 3.8 times enhanced cytotoxicity compared to free drug in Gr2000. Secondly, zebularine, a CDA inhibitor, was co-encapsulated with gemcitabine using HA-pSL. With an optimised DL of 0.95 % for zebularine, loading zebularine did not change the liposome size and zeta-potential. Compared to 3.2 folds improvement of cytotoxicity with mono-drug loaded HA-pSL than free drug, two dual-drug loaded HA-pSL showed 3.8 and 3.5 folds enhancement, respectively. Interestingly, curcumin-gemcitabine co-loaded HA-pSL could completely kill both cancer cells at high concentrations, in contrast to HA-pSL loaded with gemcitabine alone or in combination with zebularine (10 % in MIA PaCa-2 and >25 % in Gr2000 remained alive). This suggests that curcumin co-delivery may have better potential for the reversal of chemo-resistance in PC, which should be further investigated in vivo. The lack of dCK expression leads to inadequate and slow activation of gemcitabine, therefore, restoration of dCK via gene delivery may be additional approach to circumvent the resistance. Conclusions: The dual intracellular delivery approach (mediated by HA and pH-sensitivity) using HA-pSL demonstrated improved cellular uptake and in vitro cytotoxicity, but could not completely eradicate the drug-resistant Gr2000 cells. Combination therapy with curcumin, but not zebularine using HA-pSL provided advantages to overcome gemcitabine resistance with an potential to kill all drug resistant cells. Overall, this PhD study demonstrated the multifaceted nature of chemoresistance and the limitation of drug delivery with nanomedicines. Further improvement in delivery efficiency of HA-pSL to target tumours and additional manipulation of the cellular activation of gemcitabine are needed to fully tackle the chemo-resistance.