Enhanced‐mRNA Delivery Using Ultrasound‐Delivered Anchors for Bioorthogonal Ligation

Therapeutic nucleic acid delivery has many potential applications, but it remains challenging to target extrahepatic tissues in a flexible and image-guided manner. To address this issue, we report a bioorthogonal pre-targeting strategy that uses focused ultrasound (FUS) to promote the delivery of mRNA-loaded lipid nanoparticles (mRNA-LNP). We synthesized amphiphilic click reactive anchors (ACRAs) consisting of a phospholipid PEG-conjugate functionalized with trans-cyclooctene (TCO) or its companion reactive partner methyltetrazine (mTz), producing ACRA-TCO and ACRA-mTz. ACRA derivatives were screened for cellular activity, yielding functionalized DOPE-PEG (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(polyethylene glycol)) derivatives outperforming those containing saturated lipid or branched PEG. Nanobubbles encapsulating ultrasound-responsive gas delivered ACRA-TCO to targeted cells and tissues using FUS. This pre-targeting promoted the subsequent delivery of mRNA-LNP functionalized with companion ACRA-mTz. Ultrasound pre-targeting enhanced the accumulation of mTz-functionalized nanoparticles in cell cultures and mice by 75% and up to 3.6-fold, respectively, and increased gene expression using mRNA-LNP in vivo. Microbubbles loaded with ACRA roughly doubled mRNA delivery to the heart, while ultrasound alone did not. Taken together, this report presents a modular, ultrasound-enabled strategy for enhancing nucleic acid delivery in targeted tissues.