In-Situ Self-Assembling Multifunctional Peptide-Hydrogel Enables Efficient Bladder Cancer Therapy via Sustained Release of Bacillus Calmette-Guérin and Enhanced Immune Response

Intravesical instillation of bacillus Calmette-Guérin (BCG) remains the standard-of-care immunotherapy for high-risk nonmuscle-invasive bladder cancer (NMIBC). However, its clinical efficacy is frequently compromised by rapid drug washout resulting from intermittent bladder voiding, whereas intensified dosing regimens are associated with severe adverse events, including tuberculous cystitis and hematuria. Herein, we report an enzyme-responsive peptide, D-Nap-GFFYp, that undergoes alkaline phosphatase (ALP)-triggered self-assembly into a hydrogel selectively at the NMIBC tumor site. Using tumor-bearing mouse models, we demonstrate that simultaneous intravesical administration of D-Nap-GFFYp and BCG induces in situ gelation, forming a BCG-encapsulated hydrogel depot that enables sustained local release. This strategy markedly prolongs BCG retention within the bladder, thereby eliciting continuous immune stimulation characterized by enhanced M1 macrophage polarization and suppression of the pro-tumorigenic M2 phenotype. Importantly, localized hydrogel formation minimizes off-target exposure to high BCG concentrations, substantially reducing treatment-associated toxicity. Collectively, D-Nap-GFFYp functions as an effective and safe tumor-localized immune adjuvant, establishing a versatile platform for long-term BCG delivery. This work presents a rational and clinically translatable approach to improve the efficacy and biosafety of bladder cancer immunotherapy.