(W1030-05-28) Liposomal Amodiaquine for Localized Therapy of Non-Small Cell Lung Cancer (NSCLC)

Purpose: Lung cancer is a prevalent form of cancer worldwide, accounting for about 13% of all cancer cases and being the primary cause of cancer-related deaths. Non-small cell lung cancer (NSCLC) represents the most common type of lung cancer, making up 85% of cases, and is associated with a low 5-year overall survival (OS) rate of approximately 17.4%. Currently, treatment options for NSCLC encompass chemotherapy, surgical resection, radiation, or a combination of these therapies. Recent advances in these treatment options have included immunotherapy and targeted therapy, which have shown some promise in improving patient survival rates. However, these therapies continue to exhibit limited safety and efficacy, and patients often develop acquired resistance to targeted therapies, resulting in a lack of effectiveness. To address the limitations of current therapies, there is a pressing need for newer, more effective therapies for NSCLC. One potential strategy for developing new treatments is via drug repurposing, which can expedite the drug development process. In this project, we aimed to investigate the efficacy of amodiaquine (AQ), an anti-malarial drug that has previously demonstrated efficacy in different cancer types by inhibiting autophagy and inducing apoptosis in cancer cells, as an anti-NSCLC agent. Given that NSCLC is a pulmonary disorder, we also aimed to develop a lipid-based formulation that could mitigate the adverse effects of AQ and aid in localizing the drug at the target site.

Methods: AQ-loaded liposomes were developed using a conventional thin film hydration technique containing DPPC: Cholesterol (7:3) with a total lipid concentration of 10 mM.  The prepared AQ-liposomes were subjected to physicochemical characterization, including particle size, zeta potential, polydispersity index, and drug encapsulation. Solid-state characterization studies such as differential scanning calorimetry (DSC) were performed to assess the successful encapsulation of AQ in liposomes. The efficacy of AQ-loaded liposomes against NSCLC cell line A549 was determined using cell viability assay.

Results: The physicochemical characterization of AQ-loaded liposomes yielded a particle size of 135.0±30.0 nm (Fig. 1A), with a zeta potential of -2.7±1.6 mV (Fig. 1B). The resulting formulation yielded a uniform particle size distribution as evidenced by a PDI of 0.2±0.08. The encapsulation of AQ was found to be 77.4±2.1%, with a drug loading of 38.31±0.65%.  The DSC analysis showed a sharp endothermic peak of AQ at 175⁰C, portraying its melting temperature (Fig. 1C). On the other hand, the small endothermic peak was also observed in the case of blank liposomes which could be attributed to the phase transition of lipids from gel to liquid phase. Interestingly, no endothermic peak of AQ or blank liposomes was observed in AQ-loaded liposomes, which indicates successful encapsulation of AQ within the liposomal bilayer (Fig. 1C). This could also suggest a potential interaction between AQ and lipids. The cell viability assay performed in A549 cells showed a dose-dependent reduction in the cell viability when treated with AQ-loaded liposomes (IC₅₀: 17.5±0.3 mM) compared to plain AQ (IC₅₀: 37.5±0.1 mM), yielding ~2.1 fold reduction in the IC₅₀ value (Fig. 2). This enhanced efficacy of AQ is attributed to the increased permeation due to the lipid interaction with the cell membrane.

Conclusion: The present study focuses on developing an inhaled therapy of AQ for the treatment of non-small cell lung cancer. The current results revealed the successful development of AQ-loaded liposomes with < 200 nm particle size and >75% encapsulation. The cell viability assay showed significantly enhanced therapeutic efficacy against the A549 cell line compared to the plain drug. Further evaluation of AQ-loaded liposomes using other 2D and 3D in-vitro-based assays is required to establish the efficacy of liposomes in NSCLC cell lines. Moreover, since we aimed to develop an inhaled therapy, assessing the deep lung deposition potential of AQ-loaded liposomes is pivotal. To understand the dose/dosing frequency of AQ-loaded liposomes, an in-vitro study in simulated lung fluid will also be performed along with the stability study at 4⁰C and 25⁰C to determine the shelf life of the developed formulation. Lastly, mechanistic evaluation is also important in understanding the molecular pathways through which AQ inhibits tumor growth and proliferation.