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

Abstract 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.