An Elvitegravir Nanoformulation Crosses the Blood-Brain Barrier and Suppresses HIV-1 Replication in Microglia

Abstract

Even with an efficient combination of antiretroviral therapy (ART), which significantly decreases viral load in human immunodeficiency virus type 1 (HIV-1)-positive individuals, the occurrence of HIV-1-associated neurocognitive disorders (HAND) still exists. Microglia have been shown to have a significant role in HIV-1 replication in the brain and in subsequent HAND pathogenesis. However, due to the limited ability of ART drugs to cross the blood-brain barrier (BBB) after systemic administration, in addition to efflux transporter expression on microglia, the efficacy of ART drugs for viral suppression in microglia is suboptimal. Previously, we developed novel poly (lactic-co-glycolic acid) (PLGA)-based elvitegravir nanoparticles (PLGA-EVG NPs), which showed improved BBB penetration in vitro and improved viral suppression in HIV-1-infected primary macrophages, after crossing an in vitro BBB model. Our objective in the current study was to evaluate the efficacy of our PLGA-EVG NPs in an important central nervous system (CNS) HIV-1 reservoir, i.e., microglia. In this study, we evaluated the cyto-compatibility of the PLGA-EVG NPs in microglia, using an XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay and cellular morphology observation. We also studied the endocytosis pathway and the subcellular localization of PLGA NPs in microglia, using various endocytosis inhibitors and subcellular localization markers. We determined the ability of PLGA-EVG NPs to suppress HIV-1 replication in microglia, after crossing an in vitro BBB model. We also studied the drug levels in mouse plasma and brain tissue, using immunodeficient NOD scid gamma (NSG) mice, and performed a pilot study, to evaluate the efficacy of PLGA-EVG NPs on viral suppression in the CNS, using an HIV-1 encephalitic (HIVE) mouse model. From our results, the PLGA-EVG NPs showed ~100% biocompatibility with microglia, as compared to control cells. The internalization of PLGA NPs in microglia occurred through caveolae-/clathrin-mediated endocytosis. PLGA NPs can also escape from endo-lysosomal compartments and deliver the therapeutics to cells efficiently. More importantly, the PLGA-EVG NPs were able to show ~25% more viral suppression in HIV-1-infected human-monocyte-derived microglia-like cells after crossing the in vitro BBB compared to the EVG native drug, without altering BBB integrity. PLGA-EVG NPs also showed a ~two-fold higher level in mouse brain and a trend of decreasing CNS HIV-1 viral load in HIV-1-infected mice. Overall, these results help us to create a safe and efficient drug delivery method to target HIV-1 reservoirs in the CNS, for potential clinical use.

Keywords: HIV; antiretroviral therapy; elvitegravir; microglia; nanomedicine.

Figure 1

Biocompatibility of PLGA-EVG NPs in MMG. (A) The percentage of cell viability of MMG exposed with 0–20 µM test compounds include EVG, PLGA-EVG NPs, and 30% HS bound-PLGA-EVG NPs complexes (HS30@PLGA-EVG), measured by XTT assay. (B) Microscopic images of MMG with negative controls, positive control, and the highest concentration of EVG (20 µM) used in the cell viability assay. As a positive control, 5% acetonitrile was used. Mean ± SEM values are from five replicates.

Figure 2

Internalization mechanism of the PLGA-C6 NPs in MMG. (A) Confocal images of cellular uptake of PLGA NPs (green) in MMG (blue nuclei), in the presence of various endocytosis inhibitors. (B) Relative cellular uptake percentage calculated from the mean fluorescence intensity measured by flow cytometry in MMG. Mean ± SEM are from three measurements. * p < 0.05. (C) Confocal images of sub-localization of PLGA-C6 NPs (green) in MMG (blue nuclei), in the presence of early endosome, late-endosome, lysosome, and mitochondria markers (red). Cells were visualized under 400× magnification (Bar = 5 µm).

Figure 3

Viral suppression of EVG and PLGA-EVG in HIV-1-infected MMG after crossing an in vitro BBB model. (A) Experiment design to perform viral suppression study in MMG in an in vitro BBB model. (B) Daily BBB membrane integrity presented as TEER values. (C) Patterns of viral dynamics in HIV-1-infected MMG, with treatment response of EVG, PLGA-EVG, and PLGA-Blank NPs after crossing the in vitro BBB model. The p24 levels were normalized to the control MMG and reported as a percentage of the control group. (D) Treatment efficacy comparison of EVG and PLGA-EVG NPs presented as Area under the viral dynamic curve AUC_(0–t). Mean ± SEM values were graphed from triplicate samples, with MMG derived from one donor. * Indicates p < 0.05 compared to control; ^# indicates p < 0.05 compared to EVG native drug.

Figure 4

Plasma and brain concentrations of EVG and PLGA-EVG NPs in mice. (A) Plasma concentrations of EVG were measured after i.p. administration of a 20 mg/kg dose of native EVG (n = 8) or PLGA-EVG (n = 8) to mice by LC–MS/MS. Area under the curve (AUC) and maximum concentration (C_max) were analyzed, using non-compartmental analysis by PK solver. (B) Brain concentrations of EVG were measured after sacrificing mice at 96-hour time point. Mean ± SEM values were graphed from eight mice measurements for each group. * Indicates p < 0.05 compared to EVG native drug.

Figure 5

Viral suppression of EVG and PLGA-EVG in a HIVE mouse model. Viral suppression of EVG was measured after i.p. administration of a 200 mg/kg dose of vehicle control (PLGA-Blank NPs, n = 2), EVG native drug (n = 2), or PLGA-EVG (n = 2) to HIVE mice. CNS viral load was presented as HIV-gag level and normalized to the control group, which was injected i.p. with PLGA-Blank NPs, and reported as a percentage of the control group.

Figure 6

Graphic illustration representing the use of EVG nanoformulation across the BBB, to suppress the CNS HIV-1 viral replication in microglia cells.