Heat-Stressed Melanoma Exosomes Conjugated with RRG-Doxorubicin as a Prodrug Platform for Anti-Tumor Activity

Translating immunogenic cell death (ICD)-based therapies into clinical practice demands precise and biocompatible delivery platforms capable of targeted drug release. Here, we report the engineering of exosomes derived from heat-stressed B16F10 melanoma cells as a homologous, self-targeted prodrug nanoplatform for enhanced melanoma immunotherapy. The exosomal surface was functionalized via biorthogonal click chemistry with an arginine-arginine-glycine (RRG) peptide conjugated to doxorubicin (DOX), yielding RRG-DOX-modified heat-stressed exosomes (HS-Exo-RRG-DOX). The physicochemical characterization of HS-Exo-RRG-DOX confirmed optimal size, surface charge, and colloidal stability suitable for systemic delivery. Selective cytotoxicity and enhanced cellular uptake were demonstrated in B16F10 melanoma cells compared to normal murine fibroblasts (L929), underscoring the homologous targeting advantage. Furthermore, HS-Exo-RRG-DOX significantly suppressed melanoma cell migration and invasion, accompanied by downregulation of key metastasis-associated proteins. Importantly, the prodrug nature of the conjugate was validated, confirming controlled drug activation within the tumor microenvironment. Collectively, this work presents HS-Exo-RRG-DOX as a multifunctional exosomal prodrug platform that bridges ICD-mediated immune activation with targeted chemotherapy, offering a promising avenue to overcome the limitations of conventional anticancer therapeutics and advance exosome-based cancer immunotherapy.