For complexation of mRNA into polyplexes, double-pH-responsive lipo-xenopeptides (XP), comprising tetraethylene pentamino succinic acid (Stp) and lipoamino fatty acids (LAFs), were combined with PEGylated lipids, either DMG-PEG 2 kDa (DMG-PEG) or azido-group-containing DSPE-PEG 2 kDa (DSPE-PEG-N3), to increase colloidal stability and to facilitate ligand-mediated targeted mRNA delivery. LAF-XPs mixed with DMG-PEG at low (1.5% and 3%) molar ratios improved colloidal stability and retained transfection efficiency. PEGylation also enabled the formulation of otherwise unstable carrier complexes and prevented aggregation induced by salt, proteins, and serum. PEGylation of more positively charged Stp-LAF₂ mRNA polyplexes decreased fibrinogen adsorption. More neutral, LAF-rich Stp-LAF₄ polyplexes exhibited low fibrinogen binding without PEGylation. Intravenous administration of these stabilized mRNA complexes demonstrated enhanced biosafety while preserving transfection efficiency. DSPE-PEG-N3 was selected for cell targeting after strain-promoted azide-alkyne cycloaddition (SPAAC)-mediated click-coupling of DBCO-modified ligands. Higher PEG ratios (10% and 20%) provided effective shielding but reduced transfection efficiency, a drawback known as the “PEG dilemma”. Functionalization with an EGFR-targeting ligand restored transfection in EGFR-positive cell lines in a ligand-specific manner. High transfection efficiency is consistent with a lipophilic-to-hydrophilic polarity switch of LAF-XP carriers upon endosomal protonation, triggering dissociation of the PEG lipids and deshielding of the polyplex.

Synthetic polyinosinic:polycytidylic acid (poly(I:C)) offers an attractive cancer therapeutic by operating on two fronts at once, combining direct tumor cell killing with immunostimulatory activity. Yet, these dual functions can only be efficiently harnessed when intracellular delivery is sufficiently effective to enable poly(I:C) to reach and activate its intracellular receptors. We addressed this delivery challenge by developing pH-responsive formulations using lipoamino fatty acid xenopeptide (LAF-XP) carriers, composed of polar cationizable succinoyl tetraethylene pentamine (Stp) and apolar cationizable LAF building blocks in defined architectures. In particular, poly(I:C)-lipid nanoparticles (LNPs) formulated with bundle LAF₄-Stp₁ XP carriers displayed increased anti-tumoral activity at decreased dosage across multiple cancer cell models, compared to control formulations. In parallel, LAF-XP LNP-delivered poly(I:C) activated immune responses, including CXCL10 production by tumor cells, and activation of peripheral blood mononuclear cells (PBMCs), characterized by increased phenotypic markers (CD69 and LAMP-1/CD107a) and functional molecules (e.g., IFN-γ and granzyme B). Conditioned supernatant of pre-stimulated PBMCs with poly(I:C) reduced cancer cell viability, highlighting the contribution of PBMC-released factors to cancer cell death. Of particular novelty is the combination of poly(I:C) with siRNA-mediated survivin knockdown to increase apoptosis in cancer cells using the bundle LAF-XP LNP. Collectively, our findings establish efficient LAF-XP LNPs as a versatile platform that supports multi-layered therapeutic strategies.