Rational optimization of lipid nanoparticles enables efficient and low-activation mRNA delivery to platelets

Advances in gene delivery technologies have opened new avenues for modulating platelet function and expanding their applications in cell-based therapies. However, as anucleate cells, platelets have long faced the dual challenge of low transfection efficiency and susceptibility to activation during genetic manipulation. Here, we report a rationally optimized lipid nanoparticle (LNP) formulation for efficient and low-activation mRNA delivery to platelets by systematically tuning the 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol molar ratio. Five LNP formulations composed of ALC-0315, DOPC, cholesterol, and DMG-PEG₂₀₀₀ were prepared and evaluated in terms of physicochemical properties, mRNA encapsulation, platelet activation, and transfection efficiency. We found that varying the DOPC/cholesterol ratio was associated with differences in nanoparticle morphology, platelet responses, and delivery efficiency, with F3 showing the highest reporter expression among the five formulations tested. The optimized formulation enabled efficient expression of functional superoxide dismutase 2 (SOD2) mRNA in platelets without inducing significant activation. Functional studies showed that SOD2 overexpression markedly reduced mitochondrial reactive oxygen species (mtROS) and total ROS levels while preserving platelet ultrastructure and aggregation function. This study establishes a platelet-compatible LNP platform for functional mRNA delivery and provides new insights into the structure-activity relationship of LNP formulations in platelet gene delivery.