Bioactivatable conjugated nanocarriers for synergistic mRNA-mediated cancer immunotherapy

mRNA immunotherapy holds transformative potential in oncology, yet single-agent approaches frequently fail against immunosuppressive tumor microenvironments (TMEs). Bioactivatable co-delivery of immunogenic chemotherapy alongside mRNA offers mechanistically synergistic advantages, yet coformulation of hydrophobic small-molecule prodrugs with hydrophilic mRNA in a single stable nanoparticle remains a major unresolved engineering challenge. We synthesized an activatable lipid-prodrug by covalently tethering an hydrogen peroxide-responsive thioketal-oxaliplatin conjugate to dilinoleylmethyl-4-dimethylaminobutyrate via amide coupling, and formulated bioactivatable conjugated nanocarriers (BCNs) by microfluidic nanoprecipitation. Four formulations were comprehensively characterized and evaluated for mRNA expression, cellular uptake, endosomal escape, immune activation, cytotoxicity, and in vivo antitumor efficacy, TME remodeling, immune memory, and systemic safety. BCN-D (combination) exhibited a hydrodynamic diameter of 112 ± 3.5 nm (polydispersity index = 0.09), mRNA encapsulation efficiency of 91.5% ± 1.6%, reactive oxygen species -triggered drug release of 88% in tumor homogenate versus <12% in PBS, and 60-day colloidal stability. BCN-D was associated with increased expression of cGAS- stimulator of interferon genes pathway markers and prominent ICD hallmarks (calreticulin: 7.8-fold; HMGB1: 7.1-fold), Th1-skewed cytokine secretion (IFN-γ: 8.9-fold; IL-2: 11.3-fold), and a selectivity index (SI) >100 vs normal cells. In the B16-F10 syngeneic model, BCN-D achieved 84.7% tumor growth inhibition (combination index = 0.62), significantly prolonged survival (median not reached at day 40), and 100% durable tumor rejection upon rechallenge in complete responders. Tumor-infiltrating CD8⁺/Treg ratio improved 23-fold versus vehicle. Together, these findings characterize BCN-D as a combination nanocarrier in this preclinical model, with observed associations among ROS-responsive prodrug release, immune-marker changes, TME remodeling, and antitumor activity. These findings support further mechanistic investigation and evaluation in additional preclinical models before conclusions regarding translational applicability can be drawn. SIGNIFICANCE STATEMENT: This study presents bioactivatable conjugated nanocarriers combining reactive oxygen species-responsive oxaliplatin prodrug activation with mRNA immunotherapy, associated with altered tumor immune composition and antitumor activity in tested models. Building on prodrug-tethered ionizable lipid nanoparticles, it provides an incremental extension through thioketal-phenylboronic acid linker chemistry, an oxaliplatin prodrug designed to induce immunogenic cell death, and evaluation in melanoma. Translational relevance requires validation in additional preclinical systems.