(M1030-05-27) Charge-Tunable Synergistic Nanotherapeutics for Lung Cancer: Reactivating Tumor Suppressor p53 While Inhibiting DNA Synthesis

Purpose: Non-small cell lung cancer (NSCLC) remains the most lethal cancer among the various forms of cancer. Despite discovering various oncoprotein-targeted molecules, the 5-year survival rate has marginally improved. Moreover, development of resistance to such targeted therapy is inevitable. There is a dire need for strategy targeting root cause of oncogenesis while leveraging nanomedicine for efficiently delivering lethal combination to the tumor site. We hypothesized that reactivating tumor suppressor protein while simultaneously inhibiting DNA synthesis would be lethal to cancer cells with minimal chances of resistance development (Fig. 1). The diminished activity of p53 plays a vital role in promoting resistance to first-line anticancer agents, such as gemcitabine. Tumor suppressor protein p53 halts cancer progression before it becomes irreversible. MDM2 inhibitor – Idasanutlin (IDA) has been reported to restore the level of p53 in cancer cells. Gemcitabine Elaidate (GEM-EL) is a lipophilic prodrug of gemcitabine, can easily enter cancer cells without using hENT transporter. Co-loading/delivery of lipophilic molecule like GEM-EL and brick dust molecule like IDA has always been challenging. The objective of this study is to formulate, optimize and characterize IDA and GEM-EL loaded charge tunable liposomes (CTL) and test its anticancer efficacy using various 2D and 3D cell culture assays in lung cancer cells. Hydrophobic ion-pairing with ionizable lipid and the use of modified hydration method was explored to simultaneous loading of both the drugs. The purpose of developing charge tunable liposomes is to enhance accumulation of liposomes in solid tumors. Overexpression of sialic acid and over secretion of lactic acid make tumor cells negatively charged with low microenvironmental pH.  This is the very first research representing the combined use of IDA and GEM-EL for synergistic anticancer effects and the development of a nano-formulation targeting lung cancer.

Methods: The first part of the study is to study the cytotoxic interaction of IDA and GEM-EL and optimize the ratio with the highest synergism. Cell viability assays with combination index study were carried out A549. To address the challenges of co-delivering GEM EL and IDA, a charge tunable liposomal formulation with ionizable lipid (D-Lin-DMA) was prepared using modified hydration methods. As illustrated in Fig. 2, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), Cholesterol, DSPE-PEG-2000, GEM EL, IDA and D-Lin-DMA was in chloroform and adsorbed onto parenteral grade low-density mannitol. After chloroform evaporation, proliposomal powder was hydrated with water and subjected to nanomilling (dual centrifuge nanomill with zirconium oxide beads, 0.3mm) to reduce particle size to form unilamellar vesicles. Batches prepared using conventional thin film hydration methods showed immediate precipitation of IDA. Batches were optimized for composition using hydrodynamic diameter, zeta potential and polydispersity index (PDI), entrapment efficiency and physical stability. Drug release study was carried out using dialysis bag method at plasma pH 7.4 and tumor pH i.e. 6. Sink condition was maintained using 0.2% tween 80.

Results: Cell viability study of IDA, GEM-EL and combination of both the drug molecules in A549 cells demonstrated a significantly lower IC₅₀ compared to monotherapies. The combination index (CI) of < 0.63 for the drug confirmed the synergistic interaction. 1:1 molar ratio of both drugs is ideal for synergistic interaction. Currently, we are investigating anticancer activity of this combination using 2 lung cancer cell line with diverse mutation. Liposomal batches prepared using unsaturated phospholipid DOPC are much more stable than DPPC. Modified hydration with ionizable lipid facilitated the hydrophobic ion pairing between carboxylic acid of IDA and tertiary amine of D-Lin-DMA. Particle size of optimized the batch was 118.3±6.5 nm with nearly neutral zeta potential (-3.7) at physiological pH 7.4 and positive charge of +22 mV at tumor pH i.e. 6. HPLC analysis revealed the encapsulation efficiencies of 60-65% for both IDA and GEM-EL. Nanomilling over 30 minutes did not result in further particle size reduction or entrapment efficiency. Cryo-TEM analysis suggested uniform sized unilamellar liposomal structures. Formulation was lyophilized using trehalose and dispersibility was excellent with marginal increment in particle size. No reduction in entrapment was observed on lyophilization. Both the drugs showed sustained release, but release of IDA was much faster than GEM-EL. Further, we are testing optimized liposomes in multicellular 3D spheroids of lung cancer cells.

Conclusion: With the continuous emergence of novel anticancer agents, the strategic combination of therapeutics has become essential to maximize clinical outcomes. In this study, we developed, characterized and tested a charge tunable liposome composed of GEM-EL and IDA by leveraging the hydrophobic complexation between IDA (weakly acidic anionic) and Dlin-DMA. This formulation offers a promising platform for the co-delivery of synergistic agents, paving the way for new combination-based therapeutic strategies. Importantly, investigation for the synergistic activity of GEM-EL and IDA in NSCLC models has never been explored so far.


Fig 1. Schematic representation of mechanisms of action of Idasanutlin and Gemcitabine Elaidate in targeting lung cancer cells.


Fig 2. (A) Schematic illustration of the liposome preparation process utilizing the modified hydration method. (B) and (C) Cytotoxicity profiles comparing the effects of Idasanutlin (IDA), Gemcitabine Elaidate (GEM EL), and their combination (IDA + GEM EL) on A549 and H460 non-small cell lung cancer cell lines.


Fig 3. Graphs depicting the hydrodynamic particle size distribution (A) and zeta potential (B) of the prepared liposomes, as determined by dynamic light scattering analysis.