Enhancing cancer cell apoptosis via co-delivery of Poly (I:C) and Survivin siRNA using Lipoamino LNPs

Despite decades of research, cancer remains a major global health challenge. Conventional therapies, such as chemotherapy, often suffer from systemic toxicity and resistance, highlighting the need for more precise treatments. Nucleic acid-based therapies offer such precision by enabling gene-level interventions tailored to disease-driving mechanisms. However, their delivery is hindered by poor stability and limited membrane permeability. Among the various delivery platforms explored for nucleic acid therapeutics, lipid nanoparticles (LNPs) stand at the forefront, effectively mitigating key limitations by protecting nucleic acids and enhancing their cellular internalization. In this study, as a therapeutic strategy against cancer, we employed polyinosinic:polycytidylic acid (poly(I:C)), a synthetic dsRNA with immunostimulatory and anticancer properties. For effective delivery, we utilized LNPs formulated with novel xenopeptide (XP) carriers, which were designed by combining polar succinoyl tetraethylene pentamine (Stp) with apolar lipoamino fatty acid (LAF) motifs in bundle and U-shape topologies. Synthesized via solid-phase peptide synthesis (SPPS), they exhibit double pH-responsive behavior, thereby enabling dynamic adaptation to biological environments and facilitating endosomal escape. LNPs were prepared by rapidly mixing ethanol-dissolved ionizable XP component, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), and cholesterol with acidic citrate-buffered poly(I:C). In the initial phase, four LNP formulations containing distinct XPs, 1621 and 1755 (bundle topology), 1612 and 1716 (U-shape), were assessed for poly(I:C) delivery across different cancer cell lines. MC3 and SM-102 served as benchmarks, while Lipofectamine™ 2000/3000 and linear Polyethylenimine (L-PEI) as transfection controls. Particle size, polydispersity index (PDI), and surface charge were measured via Dynamic and Electrophoretic Light Scattering (DLS and ELS). Encapsulation and stability were confirmed by Ribogreen and gel shift assays. A549 cells were treated with varying doses of poly(I:C), and cell viability was assessed via MTT at 48 and 72 hours post-treatment. Poly (I:C)-loaded XP-LNPs induced more efficient cancer cell killing than controls, with clear dose- and time-dependent responses. To evaluate cell line-dependency, additional screenings were also conducted in U87MG and HeLa cells for 48 hours. Across all models, LNPs formulated with bundle-topology XP, particularly 1755-LNP, achieved superior functional delivery performance, outperforming U-shaped, benchmark LNPs, and transfection controls. To enhance therapeutic efficacy and overcome resistance, we shifted from monotherapy to a combination approach using siRNA targeting Survivin (an anti-apoptotic protein overexpressed in many cancers). Using 1755-LNP as the best performer, siRNA-loaded LNPs were successfully formulated. Galectin-8 recruitment confirmed robust endosomal escape, and qPCR validated gene silencing. The siRNA combination with poly(I:C) was evaluated in HeLa cells using different dose combinations, administered either simultaneously (co-treatment) or sequentially (pre-treatment with siRNA, followed by poly(I:C) treatment after 24 hours), and viability was assessed 72 h post-administration via MTT. When tested at different doses of poly(I:C) and Survivin siRNA, the combination effect in cell killing was most pronounced at lower-dose combinations, where each RNA alone showed little/no activity. Apoptosis was further investigated using Annexin V/PI flow cytometry. This approach showed not only the value of 1755-LNP as an effective delivery platform but also the enhanced therapeutic potential of the novel combination of poly(I:C) and Survivin siRNA, together forming a translationally relevant strategy for cancer treatment.