Bhalotia, A.; Nittayacharn, P.; Mehta, M.; Iyer, A.; Hutchinson, D. W.; Cheplyansky, A.; Takizawa, K.; Nidhiry, A.; Gopalakrishnan, R.; Kosmides, T.; Exner, A. A.; Karathanasis, E.

Tumors often exhibit an extracellular matrix with elevated stiffness due to excessive accumulation and crosslinking of proteins, particularly collagen. This elevated stiffness acts as a physical barrier, impeding the infiltration of immune cells and the effective delivery of various immunotherapeutic agents, such as lipid nanoparticle-based RNA therapeutics. Here, we investigate the ability of ultrasound-activated nanobubbles (US-NBs) to increase the permeability and immunogenicity of tumors. Our results show that US-NBs physically remodel the tumor tissue by decreasing its stiffness by 60% five days after a single treatment. US-NB-treated tumors display randomly oriented collagen with a 5.47-fold lower deposition compared to untreated tumors. This leads to the effective delivery and widespread distribution of lipid nanoparticles (LNPs) in the tumor. When LNPs are assisted by US-NB, they have higher gene-transfection across pan-immune cells relative to LNPs alone. Notably, US-NB enables LNPs to genetically modify T cells directly in vivo. By effectively engaging both arms of the immune system, US-NB-assisted LNPs enhance the tumor immunogenicity and infiltration of cytotoxic cells by 4-fold when compared to LNPs alone. These results indicate that gentle mechanical stimulation of the tumor using US-NB offers a promising strategy to augment the delivery and efficacy of existing immunotherapies.