Kinetically Controlled Assembly of Janus-Structured Nanobubble-Lipid Nanoparticles via Ultrasonic Micromixing

Controlling the internal architecture and morphology of lipid nanoparticles (LNPs) beyond their size remains a central challenge in nanoparticle engineering. Conventional micromixer-based assembly methods provide rapid mixing but lack the temporal resolution to manipulate the multiple competing kinetic processes that occur during LNP self-assembly. Here, we demonstrate kinetically controlled fabrication of Janus-structured surface nanobubble-lipid nanoparticles (SNB-LNPs) using an ultrasonic micromixer that enables independent tuning of three critical time scales: mixing time (t _m), bubble generation time (t _b), and LNP self-assembly time (t _a). By systematically mapping these time scales, we establish an assembly kinetic zone diagram that defines the process windows for distinct nanoparticle architectures, including spherical LNPs, bleb LNPs, and the previously inaccessible SNB-LNPs. Cryogenic transmission electron microscopy and small-angle neutron scattering confirm the Janus morphology of SNB-LNPs, featuring a gas-filled nanobubble compartment (∼17 vol % gas phase) attached to a lipid-mRNA core. The assembly outcome can be further modulated by gas supersaturation and lipid shell composition, providing additional degrees of freedom for structural control. As a proof of concept, the ultrasound-responsive SNB-LNPs demonstrate enhanced mRNA delivery both in vitro and in vivo. This work establishes a process engineering framework for fabricating multicompartment nanoparticles with nonequilibrium architectures through kinetic control in continuous-flow microreactors.