(W1130-09-61) Liver Targeting Lipid Nanoparticle for Gene Delivery by Click Reaction Modification

Purpose: Hepatitis B virus (HBV) remains a major global health challenge, with over 296 million individuals living with chronic infection. Gene-editing technologies, particularly CRISPR-based systems, offer promising therapeutic avenues. This technique could be utilized to introduce nonsense mutations into HBV cccDNA, thereby silencing viral gene expression and halting replication. However, the therapeutic potential of base editors is limited by inefficient and non-specific delivery mechanisms in vivo, which pose risks of off-target effects and systemic toxicity. To address this critical limitation, this study aims to develop a liver-targeted lipid nanoparticle (LNP) platform capable of delivering nucleic acid specifically to hepatocytes. The key innovation of this study lies in the incorporation of click chemistry for targeted LNP surface modification under mild, aqueous conditions. By conjugating N-acetylgalactosamine (GalNAc) a ligand known to bind the hepatocyte-specific asialoglycoprotein receptor (ASGPR) to the surface of LNPs, we aim to enhance the specificity of delivery to hepatocytes. In summary, the purpose of this study is to combine the precision of click chemistry with the versatility of LNPs to overcome current barriers in gene therapy for liver-associated diseases.

Methods: Ligand Synthesis: Two GalNAc-based targeting ligands were synthesized: a mono-GalNAc conjugate (MLI-01) and a tri-GalNAc conjugate (TLI-01). MLI-01 was synthesized by attaching a DBCO-functionalized linker to a GalNAc sugar via SN1 glycosylation and amide bond formation. TLI-01 utilized a branched Tris scaffold to connect three GalNAc moieties, with subsequent conjugation to a DBCO-functionalized linker via click chemistry. The identity and purity of MLI-01 and TLI-01 were confirmed by HPLC-ELSD, LC-MS, and ¹H NMR spectroscopy. LNP Formulation: LNPs were prepared using a microfluidic system (NanoAssemblr Ignite). Lipids including ionizable lipid (D-Lin-MC3-DMA), cholesterol, DSPC, and DSPE-PEG-azide were dissolved in ethanol and combined with mRNA in citrate buffer at a 3:1 aqueous to ethanol ratio. The self-assembled LNPs were dialyzed against PBS overnight at 4°C to remove organic solvents. Successful conjugation was verified by quantifying the depletion of DSPE-PEG-azide via HPLC-ELSD. Characterization: Particle size and polydispersity index (PDI) were measured using dynamic light scattering (DLS), while zeta potential was determined by electrophoretic light scattering. Encapsulation efficiency was assessed using a fluorescent RiboGreen assay. In Vitro Testing: Biocompatibility was evaluated via WST-1 assay in HepG2 cells. Cells were incubated with nLNP (non-modified), mLNP (mono-GalNAc-modified), or tLNP (tri-GalNAc-modified) formulations containing 250 ng/well of mRNA. For functional testing, EGFP mRNA transfection was quantified through fluorescence microscopy and flow cytometry. Increased fluorescence intensity indicated improved delivery and uptake by hepatocytes.

Results: The study achieved successful synthesis of two GalNAc-based targeting ligands—MLI-01 and TLI-01—functionalized with DBCO to enable click chemistry reactions. Structural verification using mass spectrometry and NMR confirmed the identity and purity of both ligands. MLI-01 achieved a yield of 73.9% with 99.0% purity, while TLI-01, due to its more complex trivalent structure, showed a lower yield of 14.5% but similarly high purity 99.1%. Post-formulation click chemistry reactions using SPAAC were conducted to attach the ligands to the azide-functionalized LNP surface. HPLC-ELSD analysis confirmed efficient conjugation, with MLI-01 achieving full surface modification within 90 minutes and TLI-01 requiring 300 minutes due to greater steric hindrance. Dynamic light scattering (DLS) analysis showed that the average particle size of unmodified LNPs (nLNP) was 131.5 nm, while mLNP and tLNP sizes were 128.7 nm and 127.7 nm respectively. Polydispersity indices (PDI) remained low across all samples, indicating uniform size distribution. Encapsulation efficiency was also well-maintained, with nLNP, mLNP, and tLNP showing encapsulation rates of 91%, 87%, and 90%, respectively. These results demonstrated that click chemistry surface modification did not compromise particle characteristics or mRNA encapsulation. The WST-1 assay revealed no cytotoxicity in HepG2 cells treated with any LNP formulation. EGFP mRNA transfection showed enhanced expression in mLNP and tLNP groups compared to nLNP. Fluorescence microscopy images displayed increased EGFP signal intensity in modified LNP-treated cells, and flow cytometry analysis quantified a 5–12% increase in transfection efficiency.

Conclusion: This study successfully developed and validated a novel lipid nanoparticle (LNP) platform for liver-targeted delivery of nucleic acids, using click chemistry-based surface modification. Two targeting ligands—mono-GalNAc (MLI-01) and tri-GalNAc (TLI-01)—were synthesized and functionalized with DBCO to enable strain-promoted alkyne-azide cycloaddition (SPAAC) with azide-bearing PEG-lipids on LNP surfaces. Importantly, the encapsulated EGFP mRNA remained stable, and there was no evidence of leakage or degradation resulting from surface modification. And functional assays revealed significantly improved mRNA delivery efficiency. Furthermore, the modularity of the system allows for future expansion to other organs by replacing the targeting ligand with tissue-specific molecules such as peptides or antibodies. In conclusion, this study presents a robust and adaptable platform for precise nucleic acid delivery, combining the benefits of LNP technology with the specificity of click chemistry. It paves the way for the development of next-generation gene therapies with enhanced efficacy, reduced off-target effects, and potential applications across a range of diseases beyond the liver.

References: [1]Y.-C. Yang, Y.-H. Chen, J.-H. Kao, C. Ching, I.-J. Liu, C.-C. Wang, C.-H. Tsai, F.-Y. Wu, C.-J. Liu, P.-J. Chen, Permanent inactivation of HBV genomes by CRISPR/Cas9-mediated non-cleavage base editing, Molecular Therapy-Nucleic Acids 20 (2020) 480-490.
[2]R. Böttger, G. Pauli, P.-H. Chao, N. Al Fayez, L. Hohenwarter, S.-D. Li, Lipid-based nanoparticle technologies for liver targeting, Advanced Drug Delivery Reviews 154-155 (2020) 79-101.
[3]Q. Wang, T.R. Chan, R. Hilgraf, V.V. Fokin, K.B. Sharpless, M. Finn, Bioconjugation by copper (I)-catalyzed azide-alkyne [3+ 2] cycloaddition, Journal of the american chemical society 125(11) (2003) 3192-3193

liver-targeting lipid nanoparticle (LNP) by click reaction. LNP will be directly modified by DBCO-galnac ligand.

The structure of mono-GalNAc conjugate (MLI-01) and a tri-GalNAc conjugate (TLI-01)

Fluorescence image of different ligand modificationLNP transfection