Poster X4 - AXL-targeted lipid nanoparticles mediated codelivery of miR-34a and miR-200c for treatment of non-small cell lung cancer with EGFR-tyrosine kinase inhibitor resistance

Introduction: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), such as osimertinib have been the first-line treatment for non-small cell lung cancer (NSCLC) patients harboring EGFR mutations. Despite the initial promising clinical responses to EGFR-TKI, the development of resistance to EGFR-TKI in the majority of patients compromises the clinical efficacy of therapy. The development of resistance primarily arises from drug tolerant cells (DTC), which survive and withstand therapy via epithelial-to-mesenchymal transition (EMT), alter the transcriptional state of specific signaling pathways and eventually develop drug resistance. To overcome EGFR-TKI resistance, we developed an AXL-targeted lipid nanoparticle platform for the co-delivery of miR-34a and miR-200c (Fig 1a), aiming to reverse drug tolerant state and restore sensitivity to EGFR-TKI, thereby preventing the development of tolerance in the early stage and solving the EGFR-TKI resistance problem when it just starts. In this formulation, miR-34a and miR-200c synergistically suppressed EMT, while the conjugated Axl aptamer enabled targeted delivery of nanoparticles to DTC. Axl-LP-miR-34a-200c exhibited robust therapeutic efficacy in both osimertinib- and erlotinib-tolerant cell lines

Materials and

Methods: The Axl aptamers conjugated, miR-34a and miR-200c co-encapsulated lipid nanoparticles (Axl-LP-miR-34a-200c) were prepared by mixing miR-34a and miR-200c with empty cationic lipid nanoparticles, followed by post-insertion of Axl aptamers on the nanoparticle surface. The size and zeta potential of Axl-LP-miR-34a-200c were measured by Zetasizer Nano ZS. The encapsulation efficiency of miR was measured by QuantiT™ RiboGreen® RNA Assay Kit. The morphology was characterized by cryo-TEM. Cellular uptake efficiency was determined by flow cytometry and confocal microscopy in both osimertinib-tolerant H1975 (H1975OR) cells and erlotinib-tolerant HCC827 (HCC827ER) cells. Cytotoxicity of Axl-LP-miR-34a-200c was evaluated using alamarBlue cell viability assay in both H1975OR cells and HCC827ER cells. The optimal dosages of miR-34a and miR-200c were determined based on the therapeutic efficacy of Axl-LP-miRs at different miR-34a/miR-200c concentrations. Following optimization, the therapeutic efficacy of Axl-LP-miR-34a-200c in osimertinib-tolerant and erlotinib-tolerant cell lines was evaluated by measuring the expression levels of AXL, epithelial markers CDH1, and mesenchymal markers ZEB1 using qRT-PCR and western blotting. The growth inhibition effect of Axl-LP-miR-34a-200c on DTCs was assessed using colony assay.

Results, Conclusions, and Discussions: Axl-LP-miR-34a-200c possessed a multilamellar structure with a size of 131.57±1.17nm and a zeta potential of +17.25±3.35mV (Fig 1a). The encapsulation efficiency of miR-34a and miR-200c was 99.58%. The cellular uptake study revealed that Axl-LP-miR-34a-200c provided ~315-fold higher delivery efficiency compared to free miR-34a and miR-200c. Dose optimization study of miR-34a and miR-200c indicated that when both miRNAs were present at a concentration of 50nM, the Axl-LP-miR-34a-200c exhibited the best therapeutic efficacy in both H1975OR cells and HCC827ER cells. Axl-LP-miR-34a-200c decreased AXL expression and reversed EMT by increasing epithelial marker CDH1 levels and decreasing mesenchymal marker ZEB1 levels (Fig 1b and 1c). Consequently, Axl-LP-miR-34a-200c enhanced the sensitivity of H1975OR cells and HCC827ER cells to osimertinib and erlotinib respectively, and significantly inhibited the growth of both cells with minimal cytotoxicity. These findings suggest that the co-delivery of miR-34a and miR-200c using Axl-targeted lipid nanoparticles represents an innovative approach to combat EGFR-TKI tolerance by reversing EMT in DTC, thereby addressing the TKI resistance issue at its origin. In the future, we will evaluate the therapeutic efficacy of Axl-LP-miR-34a-200c in ex-vivo models or in vivo orthotopic tumor models of EGFR-TKI tolerance.

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