(276c) Award Submission: Impact of Surface Chemistry on Lipid Nanoparticle Transfection and Targeted Delivery

Lipid nanoparticles (LNPs) are potent delivery vehicles for multiple nucleic acid cargos, including messenger RNA (mRNA) and plasmid DNA. These systems have been used clinically for liver-targeted delivery of nucleic acid therapeutics, and their high transfection efficacy makes them valuable for treating diseases beyond the liver, such as sickle cell disease and extrahepatic cancers. However, targeted intravenous LNP delivery to extrahepatic spaces is challenged by multiple physiological parameters. NP surfaces adsorb negatively charged serum proteins, which induce aggregation and activate rapid sequestration by immune cells [1]. In particular, LNPs adsorb apolipoprotein E (ApoE), which enhances accumulation in the liver by binding to hepatocytic low-density lipoprotein (LDL) receptors [2]. Moreover, efficient gene delivery must mitigate off-target LNP uptake by more populous cell types. LNP surfaces for targeted extrahepatic gene delivery can therefore benefit from surface modifications.

The objective of this work is to investigate the transfection efficacy of a library of LNPs at varying lipid molar compositions, layered with surface polyanions using layer-by-layer electrostatic deposition [3]. Negative surface polyelectrolytes can not only minimize off-target uptake, but also facilitate covalent attachment of moieties to improve endocytosis and intracellular trafficking [4]. Rationally chosen polyelectrolyte chemistries can also impact nanoparticle biodistribution when delivered intravenously, which is advantageous for extrahepatic delivery.

Circulating LNPs are currently stabilized in physiological conditions by the presence of surface polyethylene-glycol (PEG) conjugated lipids. However, PEGylated nanocarriers may induce immune responses and reduce the efficacy of subsequent nanocarrier dosages [5]. Therefore, electrostatically adsorbed polyelectrolytes that confer stability advantages while minimizing immunogenicity may provide a viable alternative to PEGylation.

Methods

We encapsulated either GFP-expressing mRNA or plasmid DNA in LNPs, then deposited carboxylated surface polyanions composed of either saccharides (hyaluronic acid, HA), polypeptides (poly-L-aspartate, PLD; poly-L-glutamate, PLE) or acrylic groups (polyacrylic acid, PAA). Transfection efficacy of unlayered and layered LNPs was assayed in vitro in EL4 lymphoma cells, RAW 264.7 macrophages, and HEK293T epithelial cells via flow cytometry. Layered LNP intracellular trafficking patterns were assayed via confocal microscopy studies. LNP biodistribution, transfection, and circulation times were further assessed in vivo in naïve C57BL/6 mice.

Results and Discussion

Relative to unlayered cationic LNPs, LNPs layered with polyanions exhibited diameter increases of 5 to 10 nanometers and complete surface charge reversal (Fig. 1A-D, F). Encapsulation efficiency of both mRNA and pDNA remained consistent between unlayered and layered LNPs (Fig. 1E). In vitro studies in a diverse range of immune and epithelial cell lines demonstrated correlations between lipid molar compositions and LNP transfection. Layered LNPs retained transfection capacity, transfecting at comparable or higher rates to unlayered LNPs. Furthermore, the outer polyanion of layered LNPs modulated uptake and transfection in a cell-dependent manner; HA, for example, upregulated transfection in RAW macrophages but downregulated transfection in EL4 lymphoma cells. Tested in vivo, PLE and HA layers further improved LNP hepatic and splenic transfection; HA-layered LNPs demonstrated splenic specificity (Fig. 2A-B). Surface deposition of polyanions further stabilized non-PEGylated LNPs in salt and serum conditions, while preserving transfection in vivo.

These findings suggest that LNP surface chemistry can be modulated to improve targeting and uptake, to aid in the transfection of extrahepatic tissues.

Acknowledgements: Funding was provided by the Bill and Melinda Gates Foundation and the National Science Foundation.

References

[1] Kumar et al. Chemical Reviews 121, 11527-11652 (2021).
[2] Samaridou et al. Advanced Drug Delivery Reviews 154-155, 37-63 (2020).
[3] Nabar et al. Proceedings of the National Academy of Sciences 121(11), (2024).
[4] Correa et al. ACS Nano 14(2), 2224-2237 (2020).
[5] Ju et al. ACS Nano 16(8), 11769-11780 (2022).