Intravesical Delivery of P21 <scp>mRNA</scp> –Loaded Lipid Nanoparticles as a Tumor Suppressor Replacement Therapy for Bladder Cancer

Bladder cancer is characterized by high recurrence rates and limited long-term benefit from current intravesical therapies, highlighting the need for alternative localized treatment strategies. Among tumor suppressors altered in bladder cancer, CDKN1A, which encodes the cyclin-dependent kinase inhibitor p21, is recurrently inactivated and downregulated, supporting its potential as a target for tumor suppressor replacement. Here, we developed a non-viral therapeutic strategy based on chemically modified p21 mRNA encapsulated in lipid nanoparticles (p21-LNP) for intravesical delivery. Public dataset analysis, tissue microarray staining, and cell line validation showed that p21 expression decreases during bladder cancer progression and that endogenous p21 protein levels are very low in bladder cancer cells. In vitro, synthetic p21 mRNA achieved robust nuclear p21 expression and markedly suppressed bladder cancer cell proliferation, viability, and clonogenicity. Mechanistically, p21 restoration reduced retinoblastoma protein (Rb) phosphorylation, decreased Cyclin E, Cyclin B, and proliferating cell nuclear antigen (PCNA) expression, increased γ-H2A.X accumulation, and promoted apoptosis. The resulting p21-LNP showed favorable physicochemical properties for intravesical administration. In vivo, reporter mRNA-LNP mediated strong bladder-localized protein expression with limited and transient systemic distribution. In an orthotopic bladder cancer mouse model, repeated intravesical administration of p21-LNP significantly suppressed tumor growth, restored p21 expression in bladder tissues, and preserved urothelial architecture without obvious adverse effects. Together, these findings establish intravesical delivery of p21 mRNA-LNP as a clinically compatible strategy for localized tumor suppressor replacement therapy in bladder cancer.