(W1130-03-17) Re-engineering of Lipid Nanoparticles Using Ionic Liquids for Brain Drug Delivery

Purpose: Brain endothelial cells (BECs) are one of the major constituents of the blood-brain barrier (BBB) (1). BECs, neurons and astrocytes are key cell types that maintain the health, structure and function of the integrated circuit: “neurovascular unit.” BEC damage and dysfunction drive the pathogenesis of various neurological disorders, as BECs play a crucial role in preserving the homeostatic balance of the neurovascular unit (2, 3). Thus, targeting BECs for drug delivery is a viable strategy to rescue neuronal dysfunction and maintain the overall homeostasis of the neurovascular unit. Importantly, this approach is innovative as it bypasses the need for drugs to overcome the BBB, the holy grail of brain drug delivery. Lipid nanoparticles (LNPs) have revolutionized the field of drug delivery and are carriers for macromolecules like small interfering RNA (siRNA) in the FDA-approved product, Onpattro. They are also used as mRNA carriers in the Moderna and Pfizer-BioNTech COVID-19 vaccines (4-6). However, LNPs are inherently directed to the liver post-intravenous administration and < 1% of the injected dose reaches the BECs (7, 8). To address this challenge, we propose to coat LNPs with ionic liquids (IL) to re-direct their biodistribution and increase their accumulation in the BECs. ILs are comprised of cations and anions and are a class of biomaterials with tunable structure-function dynamics (9-11). The cation-anion coordinating interactions within ILs also govern their interactions with the external microenvironment such as blood and plasma proteins. We aim to leverage and extend the physicochemical properties of ILs into LNPs to regulate their interactions with plasma proteins and red blood cells in the bloodstream. Thus, we hypothesize that re-engineering the classical LNPs via coating ILs onto LNP surfaces or incorporating into LNPs will decrease plasma protein binding and thereby, reduce non-specific accumulation in the liver and as a consequence, increase their accumulation in distant sites such as the BBB.

Methods: siRNA-loaded LNPs were formulated using C12-200, an ionizable cationic lipid, and helper lipids. siRNA-LNPs were coated with different volumes of 1:1 or 1:2 choline:trans-2-hexenoate ILs using bath sonication whereas the ILs were incorporated into the LNP monolayer during LNP synthesis for preparing IL-incorporated LNPs. Post-bath sonication, LNPs were analyzed using dynamic light scattering, and significant changes in the hydrodynamic diameters and/or zeta potential were used as determinants of IL-coating. Colloidal stability studies were performed over seven days upon interim storage of the samples at 2-8 ^oC. The morphology of IL-re-engineered LNPs was studied using TEM and cryoTEM. Serum stability was also performed to determine the effect of serum proteins/cell culture conditions on LNP particle characteristics. We also studied the total plasma protein adsorption on IL-re-engineered LNPs using the MicroBCA assay. Uptake of IL-re-engineered LNPs in mouse brain endothelial cells and mouse motor neurons was studied using fluorescence microscopy and flow cytometry.

Results: For coating LNPs with ILs, we added 12.5, 25, or 50 µL of IL to the LNPs and bath sonicated the mixture for 4, 60, and 180 minutes. A significant decrease in LNP diameters was observed post-60 minutes of bath sonication after the addition of 12.5 µL and 25 µL of IL. We also noted a significant shift in the zeta potential of LNPs post-four, -60, and -180 minutes of bath sonication after the addition of 12.5, 25, and 50 µL of IL. Significant changes in LNP diameters and surface charge after bath sonication were suggestive of IL coating. Non-significant changes were observed in the sizes and zeta potential values of uncoated LNPs and those coated with 12.5 µL over the seven-day storage regime. LNPs coated with 12.5 µL IL appeared to be spherical, similar to uncoated LNPs upon imaging with TEM and cryo-TEM. A “halo” surrounding the LNP core was seen for IL-coated LNPs, suggesting an IL-coat formation around the LNP. We observed a significant reduction in the plasma protein adsorption of LNPs coated with 12.5 µL IL compared to uncoated LNPs. The lower protein adsorption on IL-coated LNPs compared to uncoated LNPs can potentially cause reduced opsonization and clearance upon intravenous administration. Also, IL-coated LNPs can accumulate in the liver to a lesser extent since plasma protein adsorption directs bare LNPs to the liver. IL-re-engineered LNPs also increased siRNA uptake into mouse brain endothelial cells and mouse motor neurons.

Conclusion: Overall, IL-coated LNPs demonstrated colloidal stability, lower plasma protein adsorption, and increased uptake into mouse brain endothelial cells and neurons—traditionally hard-to-deliver cellular targets. Our data also indicates the potential of using ILs as a biomaterial to surface-modify LNPs to reduce surface plasma protein adsorption. Together, the preliminary studies provide proof-of-concept that traditional LNPs can be coated with choline trans-2-hexenoate IL. Using this approach, we aim to leverage the benefits offered by ILs to the clinically approved LNP platform to control their interactions in the bloodstream and redirect their biodistribution to the BBB.

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Figure 1. Schematic representation of coating LNPs with ILs using bath sonication (a) and the proposed accumulation of IL-coated LNPs in the BBB post i.v. administration (b).


Figure 2. TEM (a) and Cryo-TEM (b) micrographs of uncoated and IL-coated LNPs.


Figure 3. Cellular uptake of standard, IL-coated, and IL-incorporated LNPs containing Cy5 siRNA into bEnd.3 BECs (a) and NSC-34 motor neurons (b) using flow cytometry. bEnd.3 BECs and NSC-34 motor neurons were treated for 4 h with standard LNPs, IL-coated LNPs, IL-incorporated LNPs, and siRNA-Lipofectamine RNAiMAX complexes loaded with 50 nM Cy5 siRNA in complete growth medium or Neurobasal differentiation medium in a humidified incubator at 37 °C and 5% CO2. Lipofectamine-siRNA complexes and untreated cells were used as positive and negative controls, respectively. Data are presented as a -fold increase in Cy5 siRNA uptake (% Cy5+ cells) relative to untreated cells (n=4). Statistical analysis was done using one-way ANOVA with Dunnett’s multiple comparisons test. *p < 0.05, **p < 0.001, ****p < 0.0001.