Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) activates death-receptor pathways to trigger caspase-dependent apoptosis, yet recombinant TRAIL has demonstrated limited activity in non-small cell lung cancer (NSCLC) because of rapid clearance and apoptotic resistance. Delivering TRAIL as messenger RNA (mRNA) may overcome these limitations, but mRNA therapeutics remain limited in oncology due to delivery inefficiencies, instability, and incomplete cytosolic release. This study evaluated biodegradable cationic lipopolymers for TRAIL mRNA delivery and examined whether combining TRAIL mRNA with small interfering RNAs (siRNAs) targeting anti-apoptotic mediators could strengthen apoptotic responses in resistant NSCLC settings. Biodegradable cationic lipopolymers were screened based on their delivery profiles. Nanoparticles were characterized by hydrodynamic diameter, zeta potential, and transmission electron microscopy (TEM). A549 lung adenocarcinoma cells were used for mechanistic studies, including multi-day viability assays, caspase-3/7 activity using the Caspase-Glo assay (24-72 hours), mitochondrial membrane potential analysis using tetramethylrhodamine ethyl ester (TMRE), nuclear morphology by Hoechst staining and confocal microscopy, DNA damage assessment by alkaline comet assay, and enzyme-linked immunosorbent assay (ELISA) for TRAIL protein detection. Additional NSCLC cell lines: Calu-3, H1975 GFP+, and H1299 GFP+ were used for viability-based bioassays to support mRNA-induced TRAIL activity. The selected polymer formed compact nanoparticles with 100-200 nm size and+15-40 mV zeta-potential. In A549 cells, TRAIL mRNA induced a clear time-dependent apoptotic response: viability declined progressively and reached approximately 40-50% at later time points with higher mRNA doses. Caspase-3/7 activity increased by ∼2-fold at 24 hours and remained detectable at 48-72 hours. Confocal imaging showed mitochondrial depolarization together with condensed and fragmented nuclei, and comet assay confirmed DNA strand damage. siRNAs alone reduced viability but did not match the effect of TRAIL mRNA. In combination studies, siRNAs allowed lower TRAIL mRNA doses to achieve viability reductions comparable to higher-dose mRNA alone and maintained reduced viability further into the time course, improving both potency and duration of response. The delivery platform evaluated here supported effective TRAIL mRNA activity in A549 cells, and incorporating siRNAs further reinforced and prolonged the apoptotic response at reduced mRNA doses. These findings highlight a practical path toward combination nucleic acid strategies designed to counter apoptotic resistance in NSCLC. Portions of this abstract were drafted with the assistance of AI under the supervision and full scientific verification of the authors. Citation Format: Gurkirat Singh Sandhu, Hasan Uludag.. Polymeric delivery of TRAIL mRNA for inducing apoptosis in non-small cell lung cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3922.
Sandhu et al. (Fri,) studied this question.