Ionic Liquid Coating-Driven Nanoparticle Delivery to the Brain: Applications for NeuroHIV

Abstract

Delivering cargo to the central nervous system (CNS) remains a pharmacological challenge. For infectious diseases such as HIV, the CNS acts as a latent reservoir that is inadequately managed by systemic antiretrovirals (ARTs). ARTs thus cannot eradicate HIV, and given CNS infection, patients experience neurological deficits collectively referred to as "neuroHIV". Herein, the development of bioinspired ionic liquid-coated nanoparticles (IL-NPs) for in situ hitchhiking on red blood cells (RBCs) is reported, which enables 48% brain delivery of intracarotid arterial- infused cargo. Moreover, IL choline trans-2-hexenoate (CA2HA 1:2) demonstrates preferential accumulation in parenchymal microglia over endothelial cells post-delivery. This study further demonstrates successful loading of abacavir (ABC), an ART that is challenging to encapsulate, into IL-NPs, and verifies retention of antiviral efficacy in vitro. IL-NPs are not cytotoxic to primary human peripheral blood mononuclear cells (PBMCs) and the CA2HA 1:2 coating itself confers notable anti-viremic capacity. In addition, in vitro cell culture assays show markedly increased uptake of IL-NPs into neural cells compared to bare PLGA nanoparticles. This work debuts bioinspired ionic liquids as promising nanoparticle coatings to assist CNS biodistribution and has the potential to revolutionize the delivery of cargos (i.e., drugs, viral vectors) through compartmental barriers such as the blood-brain-barrier (BBB).

Keywords: brain delivery; cellular hitchhiking; ionic liquids; nanoparticles; red blood cells.

Figure 1

Ionic liquid coats both empty PLGA NPs and those loaded with 60 µg mL⁻¹ abacavir. Empty bare PLGA (black line) and IL‐coated PLGA (red line) NPs undergo an increase in size A) and anionic shift in surface charge B) when abacavir is loaded into Bare PLGA (blue line) and IL‐coated PLGA (green line) NPs. Scanning Electron Microscopy (SEM) of Bare PLGA and C) IL‐coated PLGA D) shows morphological changes upon IL coating. Scale = 1 µm.

Figure 2

IL‐coated PLGA NPs encapsulate abacavir (ABC), suppress viral replication in HIV‐1 treated human PBMCs without cytotoxicity, and show enhanced human microglia uptake in vitro. A) HIV‐1_BaL viral replication (n = 2) is attenuated by CA2HA 1:2‐coated PLGA NPs loaded with abacavir (ABC; 60 µg mL⁻¹; n = 3), free ABC alone (60 µg mL⁻¹) (n = 3), or CA2HA 1:2‐coated empty PLGA NPs (n = 3). ^* indicates significant difference from mock‐infected cells (n = 2); ^ indicates significant difference from HIV‐infected cells; p < 0.05 (Repeated‐Measures ANOVA). B) Bare and IL‐coated PLGA NPs with ABC (60 µg mL⁻¹) show little cytotoxicity when compared to mock‐infected PBMCs. ^* indicates significant difference from mock‐infected cells; ^ indicates significant difference from HIV‐1 infected cells; p < 0.05 (One‐Way ANOVA). C–E’) Immunocytochemistry on cultured primary human microglia : C–C’) Media‐control, D–D’) Bare PLGA NPs loaded with DiD (purple), and E–E’) IL‐PLGA NPs loaded with DiD. Cells were co‐labeled with anti‐Iba‐1 (green) and Hoechst nuclear stain (blue). Intracellular DiD (purple) accumulation was qualitatively greater when PLGA‐NPs were coated with IL (see E‐E’). Scale = 50 µm.

Figure 3

IL‐NPs dramatically enhance delivery to the brain in vivo and influence regional abacavir accumulation. A–D) Sprague‐Dawley rat brain cross‐sections shown after treatment with: (A) Saline, (B) Bare PLGA NPs loaded with DiD (purple), or (C) IL‐coated PLGA NPs loaded with DiD. Scale bar = 1 mm. (D) Signal quantified by densitometry (area × mean intensity; n = 1/group). E) Biodistribution (%) of injected DiD in isolated organs (% ID organ, n = 3/group; mean ± SEM). † denotes significant difference from respective PLGA‐DiD‐treated group; p < 0.05 (paired two‐tailed t‐test for means). F,G) Representative differences, by ¹H‐NMR spectroscopy, in abacavir (ABC) regional brain accumulation in Sprague‐Dawley rat brains (n = 3/group) post intra‐carotid injection for (F) empty IL‐PLGA NPs and (G) IL‐PLGA NPs loaded with ABC. Key proton peak for ABC presence at 8.1 ppm is indicated (see red box, panel G).

Figure 4

IL‐PLGA NPs enter the brain by shearing through blood vessels and are uptaken by microglia in the caudate/putamen. A) Bare PLGA NPs sporadically in presumed endothelial cells at the boundary of small blood vessels. B,C) IL‐PLGA NPs in parenchymal Iba‐1⁺ microglia and around blood vessels in presumed endothelial cells. C) DiD signal from IL‐coated NPs observed in every microglial soma captured in the field and in several suspected endothelial cells surrounding apparent blood vessels. D,D’) Labeling with von Willebrand factor confirming presence of DiD in endothelial cells. E) Z‐stack imaging supports intracellular localization of DiD in microglia (see bottom and right orthogonal views for virtual cross‐section). F,F’) Frequent uniform DiD co‐localization to microglia next to large vessels. ^* Indicates blood vessel. Arrows localize DiD in panels E, F, and F’. Scale bars = 10 microns in every panel.

Figure 5

IL‐PLGA DiD NPs in the brain co‐localize selectively with microglia in vivo. A–D) Co‐localization of DiD‐loaded IL‐NPs (D) with microglia (C), but not astrocytes (B), is demonstrated in the parenchyma of the rat dorsal striatum (inset shows the head of the caudate across panels). E–I) A blood vessel (outlined in E‐F) reveals DiD (I) co‐localization with microglia (H), but not astrocytes (G) (arrows localize DiD signal across panels). J–L) Somal expression of DiD is evident in microglia. M) Fractional gated representation of FACS quantification (CDllb⁺ versus CDllb⁺DiD⁺) of isolated and purified microglia shows high co‐localization with IL‐ PLGA DiD NPs (blue) versus saline background (green), the latter of which was only CDllb⁺ (n = 3 internal repetitions of n = 1 brain extract/group ± standard deviation). CC = corpus callosum, LV = lateral ventricle, AC = anterior commissure. Scale bar = 1 mm in Panels A‐D, 50 microns in Panels E‐I, 10 microns in Panels (J–L).