(I-351) Impact of Surface Chemistry on Lipid Nanoparticle Transfection and Targeted Delivery

Introduction:: Lipid nanoparticles (LNPs) are potent transfection agents for multiple nucleic acid cargos, including mRNA and plasmid DNA. LNPs can therefore mediate in vivo therapeutic delivery to the bone marrow, which expresses genetic diseases such as sickle cell disease and hemophilia. However, 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. Negative surface polyelectrolytes can not only minimize off-target uptake, but also facilitate covalent attachment of moieties to improve endocytosis and intracellular trafficking [3]. Rationally chosen polyelectrolyte chemistries can also impact nanoparticle biodistribution when delivered intravenously, which is advantageous for extrahepatic delivery.


Materials and Methods:: We encapsulated either GFP-expressing mRNA or plasmid DNA in LNPs, then deposited carboxylated surface polyanions composed of either saccharides, polypeptides or acrylic groups. Polyanions were further modified with reactive handles to enable attachment of antibodies to improve uptake and targeting. Transfection potential 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. Biodistribution, transfection, and circulation were further assessed in vivo in naïve mice.


Results, Conclusions, and Discussions:: Relative to unlayered cationic LNPs, LNPs layered with polyanions exhibited diameter increases of 5 to 10 nanometers and complete surface charge reversal. Encapsulation efficiency of both mRNA and pDNA remained consistent between unlayered and layered LNPs. In vitro studies in a diverse range of 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; polysaccharides, for example, upregulated transfection in RAW macrophages but downregulated transfection in EL4 lymphoma cells. Tested in vivo, layered LNPs demonstrated extended circulation times over unlayered LNPs; polypeptide-layered LNPs further improved splenic transfection. These findings suggest that LNP surface chemistry can be modulated to improve targeting and uptake, to aid in the transfection of extrahepatic tissues.


Acknowledgements (Optional): :

References (Optional): : [1] Kumar et al. Chemical Reviews 121, 11527-11652 (2021).

[2] Samaridou et al. Advanced Drug Delivery Reviews 154-155, 37-63 (2020).

[3] Correa et al. ACS Nano 14(2), 2224-2237 (2020).