Poster CC5 - Characterization of mRNA-LNP diffusion in fresh human cervicovaginal mucus

Introduction: Delivering proteins to the mucosal epithelia is promising to treat various diseases. Mucus is a continuously secreted gel network composed of mucins and cells, forming the first line of defense against the entry of pathogens or therapeutic particles (1, 2). mRNA is an efficient and safe way to introduce proteins intracellularly. Lipid nanoparticles (LNPs) are powerful tools for the in-vivo delivery of mRNA, which also demonstrated safety and efficacy in humans. However, the capability of mRNA-LNPs to penetrate human mucus remains unexplored, which is needed to develop effective mRNA therapeutics targeting mucosal epithelia.

Materials and

Methods: Cervicovaginal mucus (CVM) samples were obtained from donors of reproductive age with healthy vaginal microbiota following protocols approved by the Institutional Review Board of the Johns Hopkins University (IRB# NA_00085130). LNPs were prepared using manual or microfluidic mixing. Nanoparticle (NP) size and ζ-potential were measured using a Zetasizser. Encapsulation ratio was evaluated using the Ribogreen assay. Lipid envelopes and mRNA were labeled by DiD and Cy5, respectively. Multiple-particle tracking analysis was carried out using a ZEISS epifluorescence microscope equipped with 100× oil-immersion objective. Trajectories of n=130 (median) particles were analyzed in each measurement, and the coordinates of NP centroids were transformed into time-averaged mean square displacements (MSD).

Results, Conclusions, and Discussions: Results and

Discussion:
Microscopic imaging showed that mRNA-LNPs were rapidly sequestered by shed epithelial cells in CVM. In the cell-free portion of mucus, LNPs exhibited rapid diffusion as measured by MSD (timescale 1 sec) that was ~200 times higher than that of conventional carboxylate functionalized polymeric NPs of the same size. LNPs remained intact upon dilution in mucus, which was confirmed by the observation that lipids (DiD) and mRNA (Cy5) exhibited the same MSD profiles. Additionally, mRNA remained encapsulated inside the LNPs even after incubation in simulated vaginal fluids for a period of up to 2 hours.

Conclusion:
Overcoming mucus is the first step to deliver mRNA efficiently to the epithelium of the female reproductive tract. Our findings suggest that LNPs can be mucus-penetrating in human CVM. The relevance of LNP localization in shed epithelial cells requires further investigation.

Acknowledgements (Optional):

References:
(1) Cone RA. Adv Drug Deliv Rev. 2009 Feb 27;61(2):75-85.
(2) Lai SK et al. Adv Drug Deliv Rev. 2009 Feb 27;61(2):158-71.

Funding:
R01HD103124 and RO1 HD108905 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development