Abstract
Aims
Extracellular vesicles (EVs) hold great therapeutic promise, but their rapid clearance via the mononuclear phagocyte system, especially in the liver and spleen, limits clinical application. This project aims to improve EV pharmacokinetics and targeting by developing a biocompatible red blood cell (RBC) hitchhiking system.
Methods
Due to the negative charge on both EVs and RBC membranes, repulsion hinders their binding. Therefore, we engineered positively charged phosphatidylserine (PS)-binding peptides that electrostatically bind RBC membranes and attach to EVs via PS domains, forming RBC-hitchhiked EVs (RH-EVs). Upon systemic administration, RH-EVs preferentially accumulate in the lungs—the first capillary bed encountered—where RBC deformation releases EVs under shear stress. We loaded RH-EVs with a KRAS-targeting antisense oligonucleotide (ASO) and an immunomodulatory RNA (immRNA) that activates retinoic acid-inducible gene I (RIG-I), previously shown to synergistically induce immunogenic tumor death. We evaluated RH-EVs for hitchhiking efficiency, biodistribution, pharmacokinetics, safety, and anti-cancer activity in a mouse model of pancreatic cancer lung metastasis.
Results
RH-EVs demonstrated >28-fold lung accumulation at 1 hour and >10-fold retention at 24 hours compared to free EVs. They are preferentially uptaken by tumor cells and evaded immune clearance. In treated mice, RH-EVs significantly suppressed tumor growth and extended survival through robust RIG-I signaling and enhanced recruitment of macrophages, dendritic cells, CD4+/CD8+ T cells, and memory T cells. No systemic toxicity was observed across treatment groups.
Conclusion
Our EV-RBC hitchhiking strategy offers a safe, effective lung-targeted delivery platform with strong translational potential for treating lung cancer and other pulmonary diseases.