Intracellular Trafficking Kinetics of Nucleic Acid Escape from Lipid
Nanoparticles via Fluorescence Imaging
Authors: Bailey-Hytholt Christina M. , Ulinski Gregory , Dugas Julia , Haines Mohammed , Lazebnik Mihael , Piepenhagen Peter , Zarraga Isidro E. , Bandekar Amey 
Published: April 2023

Background/Introduction:
Lipid nanoparticles (LNPs) are one of the most clinically
advanced candidates for delivering nucleic acids to target cell populations, such as hepatocytes.
Once LNPs are endocytosed, they must release their nucleic acid cargo into the cell cytoplasm. For
delivering messenger RNA (mRNA), delivery into the cytosol is sufficient; however, for delivering
DNA, there is an added diffusional barrier needed to facilitate nuclear uptake for transcription
and therapeutic effect.

Objective:
The objective of the presented study was to use fluorescence microscopy to identify
nucleic acid localization to endosomes and lysosomes after escape from lipid nanoparticles in
order to optimize lipid nanoparticle formulations.

Methods:
Here, we use fluorescence microscopy to investigate the intracellular fate of different
LNP formulations to determine the kinetics of localization to endosomes and lysosomes. LNPs
used in the studies were prepared via self-assembly using a NanoAssemblr for microfluidic mixing.
As the content of polyethylene glycol (PEG) within the LNP formulation influences cellular
uptake by hepatocyte cells, the content and hydrocarbon chain length within the formulation were
assessed for their impact on intracellular trafficking. Standard LNPs were then formed using three
commercially available ionizable lipids, Dlin-MC3-DMA (MC3), Dlin-KC2-DMA (KC2), and
SS-OP. Plasmid DNA (pDNA) and mRNA were used, more specifically with a mixture of Cyanine
3 (Cy3)-labeled and green fluorescence protein (GFP) producing plasmid DNA (pDNA) as
well as Cy5-labeled GFP producing mRNA. After formulation, LNPs were characterized for the
encapsulation efficiency of the nucleic acid, hydrodynamic diameter, polydispersity, and zeta potential.
All standard LNPs were ~100 nm in diameter and had neutral surface charge. All LNPs
resulted in encapsulation efficiency greater than 70%. Confocal fluorescence microscopy was used
for the intracellular trafficking studies, where LNPs were incubated with HuH-7 hepatocyte cells
at times ranging from 0-48 h. The cells were antibody-stained for subcellular components, including
nuclei, endosomes, and lysosomes.

Results:
Analysis was performed to quantify localization of pDNA to the endosomes and lysosomes.
LNPs with 1.5 mol% PEG and a hydrocarbon chain C14 resulted in optimal endosomal
escape and GFP production. Results from this study demonstrate that a higher percentage of C14
PEG leads to smaller LNPs with limited available phospholipid binding area for ApoE, resulting in
decreased cellular uptake. We observed differences in the localization kinetics depending on the
LNP formulation type for SS-OP, KC2, and MC3 ionizable lipids. The results also demonstrate the
technique across different nucleic acid types, where mRNA resulted in more rapid and uniform
GFP production compared to pDNA delivery.

Conclusion:
Here, we demonstrated the ability to track uptake and the sub-cellular fate of LNPs
containing pDNA and mRNA, enabling improved screening prior to in vivo studies which would
aid in formulation optimization.