Precision mRNA Delivery Across Gut Epithelial Barrier via APN-Targeted Nanobody-LNP Platform

The potential of mRNA therapy in vaccines, immunotherapy, protein replacement therapy, and gene editing has been clinically validated. However, the precise delivery of mRNA to target cells remains a critical bottleneck limiting its efficacy and safety. Recently, a team from Ghent University in Belgium published a study titled "Cell-specific mRNA delivery via nanobody-functionalized lipid nanoparticles" in the Journal of Controlled Release, proposing a novel lipid nanoparticle (mRNA-LNP) solution. This approach targets aminopeptidase N (APN) on the surface of intestinal epithelial cells using nanobody-functionalized LNPs, addressing both the issue of cell-specific mRNA delivery and overcoming the intestinal epithelial barrier.

The research team focused on APN, which is highly expressed on intestinal epithelial cells. Using genetically engineered E. coli, they produced an APN-specific nanobody (VHH-AzF) carrying the non-canonical amino acid azidophenylalanine. This nanobody not only stably binds to APN but can also be chemically conjugated to LNPs. To avoid LNP aggregation caused by direct conjugation, the team employed a two-step click chemistry method (SPAAC followed by IEDDA) to precisely attach VHH-AzF to TCO-modified LNPs containing DSPE-PEG2000-TCO. The resulting APN-targeted LNPs maintained stable physicochemical properties, including particle size, polydispersity index, and zeta potential. ELISA further confirmed that the modified LNPs retained their ability to specifically bind APN.

In cell experiments, researchers incubated APN-targeted LNPs with APN-expressing cells (BHK-APN and porcine intestinal epithelial IPEC-J2-APN cells). The results showed that targeted LNPs exhibited significantly higher cellular uptake efficiency compared to unmodified or control nanobody-modified LNPs. Additionally, they effectively facilitated the translation of encapsulated eGFP mRNA into green fluorescent protein. Among these, BHK-APN cells displayed higher GFP expression levels due to more efficient endosomal acidification. Low-temperature inhibition experiments further confirmed that the uptake of targeted LNPs depended on APN-mediated specific binding rather than non-specific endocytosis.

Figure 1. Binding and Uptake of APN-Targeted VHH-LNPs by an APN-Expressing Cell Line. (Chen L, et al., 2025)

In a more physiologically relevant porcine apical-out small intestinal organoid model, APN-targeted LNPs demonstrated superior trans-epithelial delivery capabilities. Using fluorescence lifetime imaging microscopy (FLIM), the researchers observed that targeted LNPs were not only taken up by intestinal epithelial cells but also crossed the epithelial barrier via transcytosis, ultimately distributing to the central region of the organoids. Clear GFP fluorescence signals were detected, confirming effective mRNA delivery and translation in intestinal epithelial cells. In contrast, control LNPs showed only minimal distribution on the epithelial surface without trans-barrier transport.

To validate the in vivo efficacy, the team conducted a pig intestinal loop ligation experiment, injecting DiD-labeled APN-targeted LNPs into ligated jejunal loops. After six hours, the targeted LNPs were detected in the intestinal villi epithelial cells and even transported to underlying immune cells. GFP expression was observed in both epithelial and subepithelial cells. Notably, fluorescent signals and GFP expression were also detected in the drained mesenteric lymph nodes, whereas control LNPs showed almost no distribution or expression in intestinal tissues or lymph nodes.

Figure 2. αAPN-VHH-LNPs mediate targeted delivery of mRNA to the small intestinal epithelium and mesenteric lymph nodes (MLNs) in vivo. (Chen L, et al., 2025)

The core value of this study lies in its demonstration that nanobody-functionalized LNPs not only enable precise mRNA delivery to APN-expressing intestinal epithelial cells but also, for the first time, confirm that this targeting system can cross the intestinal epithelial barrier and achieve effective mRNA expression in vivo. This platform is programmable-while this study focused on porcine APN, simply replacing the nanobody could adapt it to different species' target molecules. It provides a novel technical pathway for oral mRNA vaccines and mRNA-based treatments for intestinal diseases while also opening new directions for mRNA precision delivery in cancer immunotherapy and protein replacement therapy.

Currently, precise mRNA delivery remains a major challenge in the field. This study not only addresses a key obstacle in intestinal delivery but also lays the foundation for developing personalized, cell-specific mRNA therapies. With further optimization of lipid composition and mRNA types, this platform holds promise for broader clinical applications, bringing mRNA therapy closer to the goal of "on-demand delivery."

Reference

Chen L, et al. Cell-specific mRNA delivery via nanobody-functionalized lipid nanoparticles. Journal of Controlled Release, 2025: 114365.