Poster X7 - Enhancing Endosomal Escape of mRNA Delivered via Lipid Nanoparticles Using Mechanical Vibration

Introduction: Recent advancements in messenger RNA (mRNA) and lipid nanoparticle (LNP) technologies have opened up new possibilities for treating human diseases. However, a significant hurdle persists: the inefficient delivery of mRNA molecules into the cytoplasm of target cells, where the synthesis of therapeutic proteins is supposed to happen. Studies show that a substantial portion of the mRNA delivered using LNPs (nearly 98%) ends up trapped and eliminated through the endo-lysosomal pathway[1]. In response to this challenge, we present a paradigm-shifting approach that harnesses mechanical vibration to prompt endosomal escape for enhanced intracellular mRNA delivery.

Materials and

Methods: We formulated ionizable DLin-MC3-DMA (MC3) LNPs incorporated with fluorescent tracers (DiI). The LNPs were introduced into in vitro-cultured fallopian tube non-ciliated epithelial (FNE) cells. Subsequently, the cells were exposed to vibration of 65 Hz for 5 min (Fig. 1). The downstream effects of the mechanical stimulation on endosomal escape were assessed by LysoTracker deep red (LT-Red) staining and calcein release assay. Furthermore, effects of the vibration on intracellular mRNA delivery were studied by monitoring expression of GFP-encoding mRNA. Biosafety of this strategy was also evaluated using live/dead viability assay.

Results, Conclusions, and Discussions: LysoTracker staining demonstrated that the application of vibration resulted in reduced LysoTracker colocalization, indicating enhanced endosomal escape due to vibration exposure (Fig. 2A). Pearson’s correlation coefficient (PCC) was calculated to determine the endosomal escape efficiency of each treatment. Without vibration, the PCC was approximately 0.60 ± 0.02, whereas the group with vibration showed a PCC of 0.46 ± 0.12. These results suggest an endosomal escape efficiency of approximately 40% for MC3 LNPs alone and 54% when combined with vibration. Consistently, the calcein release assay revealed a more diffused calcein signal (2.63-fold increase) in the cytoplasm of cells treated with vibration, providing further evidence of improved endosomal escape triggered by vibration (Fig. 2B). Additionally, mRNA expression experiments showed a 1.67-fold increase in GFP signals in cells treated with vibration, highlighting the ability of mechanical vibration to enhance mRNA expression delivered by ionizable LNPs (Fig. 2C). Furthermore, cellular viability experiments indicated that cell viability remained high at 99.3% after treatment with LNPs and vibration, demonstrating that the mechanical stimulus did not impair cell viability (Fig. 2D).

Our research highlights the potential of biomechanically facilitated mRNA delivery to improve mRNA therapeutics. Mechanical vibration enhances endosomal escape of mRNA in ionizable LNPs, resulting in more efficient mRNA expression. In vitro experiments confirmed the safety of this approach. Thus, our work promises applications in research and clinical fields by improving the efficiency of LNP-based drug delivery.

Although numerous studies have made significant progress in synthesizing new LNPs to improve endosomal escape efficacy, these approaches often involve complex synthesis processes and raise safety concerns. In contrast, our strategy utilizes an external mechanical stimulus to boost LNP-based delivery efficiency, which can be easily implemented both in vitro and in vivo without causing potential toxicity to cells, thereby offering greater potential for clinical translation. The underlying mechanism for this strategy likely involves enhanced fusion between LNPs and the endosomal membrane, as well as amplified disruption of the LNP and endosome induced by the vibration, leading to more efficient release of the mRNA being carried.

Acknowledgements (Optional): The authors would like to thank Dr. Tsengming (Alex) Chou for his assistance with imaging and Dr. Marcin Iwanicki for the FNE cells. The work is supported by National Science Foundation (Career Award No. 2143620), National Institutes of Health (P41 EB027062), and Cystic Fibrosis Foundation (Pioneer grant VUNJAK23XX0).