ABSTRACT
The unprecedented success of mRNA-lipid nanoparticles (LNPs) has highlighted their power for protein expression, but the hours-long half-life of mRNA severely limits their use in chronic diseases. In contrast, DNA LNPs display months-long expression and genetically encode cell type specificity, but their use has been hindered by poor protein expression (orders of magnitude lower than mRNA LNPs). To overcome this, we introduce multi-stage mixing (MSM) microfluidics to control the internal structure of LNPs and use it to create core-then-shell (CTS) structured DNA LNPs. CTS LNPs display distinct thermal transitions and internal organization compared to the amorphous structure of conventional LNPs. CTS improves transfection by three orders of magnitude in vitro , outperforming gold standard reagents in hard-to-transfect cells like primary neurons. In vivo , CTS DNA LNPs augment expression by two orders of magnitude, achieving peak expression levels comparable to mRNA LNPs, but with prolonged expression. This work demonstrates how microfluidic control over nanoparticle assembly kinetics can access otherwise unattainable particle architectures, advancing both materials science and therapeutic applications.
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