Cellular
membranes are, in general, impermeable to macromolecules (herein referred
to as macrodrugs, e.g., recombinant protein, expression plasmids,
or mRNA), which is a major barrier for clinical translation of macrodrug-based
therapies. Encapsulation of macromolecules in lipid nanoparticles
(LNPs) can protect the therapeutic agent during transport through
the body and facilitate the intracellular delivery via a fusion-based
pathway. Furthermore, designing LNPs responsive to stimuli can make
their delivery more localized, thus limiting the side effects. However,
the principles and criteria for designing such nanoparticles remain
unclear. We show that the thermodynamic state of the lipid membrane
of the nanoparticle is a key design principle for acoustically responsive
fusogenic nanoparticles. We have optimized a cationic LNP (designated
LNPLH) with two different phase transitions near physiological
conditions for delivering mRNA. A bicistronic mRNA encoding a single
domain intracellular antibody fragment and green fluorescent protein
(GFP) was introduced into a range of human cancer cell types using
LNPLH, and the protein expression was measured via fluorescence
corresponding to the GFP expression. The LNPLH/mRNA complex
demonstrated low toxicity and high delivery, which was significantly
enhanced when the transfection occurred in the presence of acoustic
shock waves. The results suggest that the thermodynamic state of LNPs
provides an important criterion for stimulus responsive fusogenic
nanoparticles to deliver macrodrugs to the inside of cells.