Purpose: Non-small-cell lung cancer (NSCLC) accounts for 80-85% of all lung cancers and is the significant cause of cancer deaths all over the world. There is growing evidence that cancer cells adapt modified autophagy mechanisms to counter either chemotherapeutic agents or environmental toxins, and exploit these pathways for a suitable tumor environment and to develop chemo-resistance. Hence, there is a need to identify newer drugs showing an anticancer activity through modulation of autophagy. Amodiaquine, an FDA approved antimalarial has been reported as a modest autophagy inhibitor, and is shown to work by blocking autophagy mediated lysosomal function. However, higher dose and significant hepatotoxicity limits further exploration and potential clinical use of amodiaquine as a stand-alone/combinatorial anticancer agent. In the project, we hypothesize that amodiaquine’s modest anticancer efficacy is linked with moderate cellular uptake of drug, which will be improved by enhancing intracellular accumulation of the drug. In this project we aim to develop biodegradable PLGA nanoparticles of amodiaquine through a scalable high pressure homogenization method and to determine their efficacy to treat NSCLC.
Methods: Amodiaquine-loaded PLGA nanoparticles were formulated using high pressure homogenizer (Nano DeBee), following optimization of formulation composition and process variables. The formulation variables included ratio of amount of PLGA502 to drug; concentration of emulsifier; components of drug solution (Pure Drug solution or Drug-Cyclodextrin complex). Briefly, a coarse emulsion was formulated using a probe homogenizer with an oil phase of PLGA solution in dichloromethane (20 mg/ml), aqueous internal phase of amodiaquine or amodiaquine-CD complex and 1% polyvinyl alcohol as an external aqueous phase with polyethylenimine (PEI).Then, it was pre-cooled followed by processing through high pressure homogenizer for 7cycles at 30,000psi pressure. The obtained emulsion was stirred at room temperature overnight for organic solvent removal followed by washing to remove unentrapped drug and excess of PVA. The final formulation was analyzed for particle size, zeta potential and drug content. Cytotoxicity potential against NSCLC was tested in 6 NSCLC cell lines; A549, H460, H157, H358, H2122 and H4006 by testing for cell viability following 72 hour drug incubation with MTT assay.
Results: The formulated amodiaquine nanoparticles were found to have uniformly sized particles with a size, polydispersity index, zeta potential and % entrapment efficiency of 299.9nm,0.212,15.8mV and 32.3% for nanoparticles without CD. As observed in cytotoxic studies (Fig. 1), amodiaquine encapsulation in PLGA nanoparticles significantly enhanced its cytotoxic potential in all 6 NSCLC cell lines with an IC50 reduction of 3-8 folds as compared to the plain amodiaquine. The IC50 values for amodiaquine decreased significantly with nanoparticles in A549 (25.6µM vs 9.6µM); H2122 (15.3 µM vs 2.0 µM); H358 (34.2 µM vs 4.8µM), H157 (21.8µM vs 15µM); H4006 (34.4µM vs 14µM) and H460 (32.6µM vs 9µM) cell lines. These results reveal the significant increase in intracellular accumulation of drug-loaded nanoparticles in cancer cells as the developed nanoparticles delivery system can deliver the drug into cells effectively due a smallerparticle size and a positive charge on their surface. Further in-vitro release studies and autophagy assays are required to ensure the significance of this approach.
Conclusion: From the results it can be concluded that amodiaquine nanoparticles formulated through high pressure homogenization could be a promising and easily scalable strategy to treat NSCLC. This approach provides an opportunity for drug repurposing which needs numerous efforts from further studies.
Vivek Gupta– Assistant Professor, St John's University