A Peptide-Based, LNP-Free mRNA Vaccine Targeting Carcinoembryonic Antigen (CEA) for Potent Colorectal Cancer Immunotherapy: Development, Structural Dynamics, and Preclinical Efficacy

Background: Colorectal cancer (CRC) continues to be a leading cause of global oncology-related deaths. While mRNA vaccines delivered via lipid nanoparticles (LNPs) have achieved clinical success, challenges regarding systemic inflammatory potential, complex multi-step manufacturing, and cold-chain dependence persist. This study explores an alternative delivery paradigm using Nona-arginine (R9), a cell-penetrating peptide, to stabilise mRNA encoding the Carcinoembryonic Antigen (CEA), providing a biocompatible, LNP-free platform for CRC immunisation. Methods: CEA-encoded mRNA was synthesised through in vitro transcription and complexed with R9 at various Nitrogen-to-Phosphate (N/P) ratios. The resulting polyplexes were characterised using Dynamic Light Scattering (DLS) and Electrophoretic Mobility Shift Assays (EMSA). Molecular docking was employed to elucidate the structural stability of the carrier-cargo interface and the binding kinetics of vaccine-induced antibodies. The therapeutic index was validated in a CT26 murine colorectal tumour model (n=10/group), assessing tumour volume reduction, survival kinetics, and the density of tumour-infiltrating lymphocytes (TILs). Results: Optimal polyplex stability was achieved at an $N/P$ ratio of 10, yielding homogenous particles (142.5 ± 4.2 nm) with a protective zeta potential of +18.6 mV. In vivo evaluation demonstrated a 65% reduction in tumour burden and an $80\%$ survival rate in vaccinated cohorts compared to 0% in control groups. This clinical efficacy was correlated with a 3-fold increase in CD8+ T-cell infiltration, a 7.6-fold upregulation of Granzyme B, and the induction of high-affinity neutralising antibodies (∆∆G = -12.4 kcal/mol) targeting critical metastatic adhesion domains. Conclusion: The R9-mRNA platform serves as a highly effective, lipid-free alternative for CRC vaccination, eliciting a sophisticated "dual-strike" immune response. By bypassing the limitations of LNPs, this strategy offers a streamlined, stable, and potent pathway for the next generation of cancer immunotherapies.