Targeting Beclin1 as an Adjunctive Therapy against HIV Using Mannosylated Polyethylenimine Nanoparticles

Abstract

Using nanoparticle-based RNA interference (RNAi), we have previously shown that silencing the host autophagic protein, Beclin1, in HIV-infected human microglia and astrocytes restricts HIV replication and its viral-associated inflammatory responses. Here, we confirmed the efficacy of Beclin1 small interfering RNA (siBeclin1) as an adjunctive antiviral and anti-inflammatory therapy in myeloid human microglia and primary human astrocytes infected with HIV, both with and without exposure to combined antiretroviral (cART) drugs. To specifically target human microglia and human astrocytes, we used a nanoparticle (NP) comprised of linear cationic polyethylenimine (PEI) conjugated with mannose (Man) and encapsulated with siBeclin1. The target specificity of the PEI-Man NP was confirmed in vitro using human neuronal and glial cells transfected with the NP encapsulated with fluorescein isothiocyanate (FITC). PEI-Man-siBeclin1 NPs were intranasally delivered to healthy C57BL/6 mice in order to report the biodistribution of siBeclin1 in different areas of the brain, measured using stem-loop RT-PCR. Postmortem brains recovered at 1-48 h post-treatment with the PEI-Man-siRNA NP showed no significant changes in the secretion of the chemokines regulated on activation, normal T cell expressed and secreted (RANTES) and monocyte chemotactic protein-1 (MCP-1) and showed significant decreases in the secretion of the cytokines interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) when compared to phosphate-buffered saline (PBS)-treated brains. Nissl staining showed minimal differences between the neuronal structures when compared to PBS-treated brains, which correlated with no adverse behavioral affects. To confirm the brain and peripheral organ distribution of PEI-siBeclin1 in living mice, we used the In vivo Imaging System (IVIS) and demonstrated a significant brain accumulation of siBeclin1 through intranasal administration.

Keywords: Beclin1; HIV; in vivo imaging system; intranasal delivery; polyethylenimine nanoparticle.

Figure 1

Quantitative measures of viral replication and inflammatory molecules in glia. HIV replication in human microglia was measured using HIV p24 Gag protein ELISA (A). Values were determined from standard curves and are presented as the mean ± the standard error of mean (SEM) of three independent experiments. Corresponding cell culture supernatants from human microglia (B) and human astrocytes (C) were used to detect the levels of monocyte chemotactic protein-1 (MCP-1), interleukin 6 (IL-6), and regulated on activation, normal T cell expressed and secreted (RANTES) by ELISA (B,C). Values were determined from standard curves and are presented as the mean ± the SEM of three independent experiments (p < 0.05; ** vs. media, $ vs. HIV, and # vs. respective antiretroviral).

Figure 2

Target specificity in glial cells expressing mannose receptors. Representative immunofluorescent images of human microglia, human astrocytes, and human neurons transfected with polyethylenimine-mannose-small interfering fluorescein isothiocyanate (PEI-Man-siFITC) (green) and immunolabeled with the antibody against ionized calcium-binding adaptor molecule 1 (Iba1) (red, left panel), glial fibrillary acidic protein (GFAP) (red, middle panel), and microtubule-associated protein 2 (MAP2) (red, right panel), respectively, after 24 h (top panel) and 120 h (bottom panel) of transfection. 4′,6-Diamidino-2-phenylindole (DAPI) was used to label nuclear DNA (blue) (A). Representative immunofluorescent images of human microglia (green, top panel), human astrocytes (green, middle panel), and human neurons (green, bottom panel) immunolabeled with antibodies against Iba1, GFAP, and MAP2, respectively, and an antibody against the mannose receptor (red, middle panel). 4′,6-Diamidino-2-phenylindole (DAPI) was used to label nuclear DNA (blue) (B). Images were acquired using an inverted fluorescence microscope with a 560 Axiovision camera using 40× magnification (Zeiss). Individual neurons were assessed for survival using time-lapse imaging at the indicated time points following the indicated treatments over 72 h (C). Neuronal viability was confirmed using a live/dead cell fluorescence assay and manually quantified following the indicated treatments at 24, 48, and 72 h. Viability was measured by fluorescence after 120 h post-treatments (D). Error bars show the SEM for three independent experiments, with at least 50 cells per experiment.

Figure 3

Quantitative measurements of small interfering RNA (siRNA) after intranasal delivery to C57BL/6 mice. After administration with PEI-Man-small interfering Beclin1 (siBeclin1) at 10 μg (A) and 20 μg (B), and at indicated time points, postmortem brain regions were minced, and RNA was used to measure siBeclin1 concentrations by stem-loop RT-PCR. The results are expressed in concentrations (nmol/g). Values were determined from the siBeclin1 standard curves and are presented as the mean ± the SEM of three independent experiments. (p < 0.05; * vs. 1 h, # vs. 4 h, $ vs. 24 h, & vs. 48 h, and @ vs. 72 h).

Figure 4

Histological and biochemical analyses of postmortem brain regions after the intranasal delivery of PEI-Man-siBeclin1 in C57BL/6 mice. Representative images of the Nissl staining of adult mice brains removed postmortem after treatment with phosphate-buffered saline (PBS) (A,B, top panel) or PEI-Man-siBeclin1 at 10 μg (A) and 20 μg (B) at 4, 48, and 120 h post-treatment. Corresponding tissues at 1, 4, 24, and 48 h were minced and used to detect RANTES, MCP-1, tumor necrosis factor alpha (TNF-α), and IL-6 by ELISA (C,D). (A,B) Images were acquired using an inverted fluorescence microscope with a 560 Axiovision camera using 20× and 40× magnification (Zeiss). (C,D) Values were determined from the standard curves and are presented as the mean ± the SEM of three independent experiments (p < 0.05; * vs. PBS and # vs. 10 µg).

Figure 5

Live imaging analysis of DIR-labeled nanoparticles after intranasal administration in C57BL/6 mice. Liposomes labeled with a fluorescent hydrophobic dye, DIR, were injected into C57BL/6 mice intranasally alone (A), or loaded with siRNA (B), or loaded with the PEI-siRNA complex (C) (3 × 10¹² particles/mL), and the brain accumulation of liposome-siRNA formulations was imaged by the In Vivo Imaging System (IVIS) up to 96 h (D). Fluorescent accumulation was quantified in different organs recovered at necropsy (E,F).