Nanoparticle based galectin-1 gene silencing, implications in methamphetamine regulation of HIV-1 infection in monocyte derived macrophages

Abstract

Galectin-1, an adhesion molecule, is expressed in macrophages and implicated in human immunodeficiency virus (HIV-1) viral adsorption. In this study, we investigated the effects of methamphetamine on galectin-1 production in human monocyte derived macrophages (MDM) and the role of galectin-1 in methamphetamine potentiation of HIV-1 infection. Herein we show that levels of galectin-1 gene and protein expression are significantly increased by methamphetamine. Furthermore, concomitant incubation of MDM with galectin-1 and methamphetamine facilitates HIV-1 infection compared to galectin-1 alone or methamphetamine alone. We utilized a nanotechnology approach that uses gold nanorod (GNR)-galectin-1 siRNA complexes (nanoplexes) to inhibit gene expression for galectin-1. Nanoplexes significantly silenced gene expression for galectin-1 and reversed the effects of methamphetamine on galectin-1 gene expression. Moreover, the effects of methamphetamine on HIV-1 infection were attenuated in the presence of the nanoplex in MDM.

Fig. 1

Methamphetamine regulates galectin-1 gene and protein expression in MDM. MDM were incubated with methamphetamine (1, 5, 10, 50, 100 μM), for 2, 4, 6, 24 or 48 h. a Gene expression for galectin-1 was determined using Q-PCR, (n=4). Data are expressed as transcript accumulation index (2–(ΔΔCT)) or TAI. b) Galectin-1 protein expression was determined using western blotting (n=4). Immunore-active protein bands were semi-quantified by densitometric analysis. Data are expressed as relative protein levels. Representative western blot for both galectin-1 and β-actin are shown in inset. c) Representative confocal image of galectin-1 protein expression (red) following methamphetamine (10 μM) incubation (a) control (b) 24 methamphetamine (c) 48 h methamphetamine with DAPI nuclear staining (blue). Relative fluorescent units (RFU) are shown in each panel (n=4, scale bar=75.49 μM). d) Supernatants were assayed for galectin-1 protein using ELISA (n=3). Data are expressed as relative protein units, with control value set at 0. Control cells release 120±5.2 pg/ml. All statistical significance was calculated using ANOVA followed by Bonferroni post-hoc test, * p<0.001; # p<0.05. • 2 h; ■ 4 h; ▲ 6 h; ◇ 24 h; X 48 h

Fig. 2

Cellular uptake of nanoplexes. MDM were transfected with GNRs, free siRNA and nanoplexes for various time frames. a Representative Transmission electron microscopy (TEM) image of nanoplex. b The fluorescence intensity from cellular lysates was measured in untransfected, GNR alone, free siRNA^FAM alone or nanoplex treated MDM 4 h post-transfection. PL = Photoluminescence. Statistical significance was calculated using ANOVA followed by Bonferroni post-hoc test, * compared to control (n=4). c Representative 40X dark field image of MDM 24 h post transfection with nanoplexes (gold) with DAPI nuclear staining (blue) (a) and the corresponding fluorescent image (b) (scale bar=100 μM). d Representative dark-field images (100X) (a-d) with DAPI nuclear staining (blue) and corresponding fluorescent images (e-h) of untransfected (a, e), GNRs alone (b, f), free galectin-siRNA^FAM (c, g) and nanoplexes (d, h) 4 h-post transfection (scale bar=10 μM)

Fig. 3

Effect of nanoplexes on galectin-1 expression. MDM were transfected with nanoplexes for various time frames, phagocytosis activity, gene expression and protein expression were subsequently investigated. a Phagocytosis activity as determined by Zymosan Substrate CytoSelect™ 96-Well Phagocytosis Assay. Cyt D = Cytochalasin D (positive control, phagocytosis inhibitor). Data are presented as percent change in phagocytosis activity (n=4). b Gene expression for galectin-1 using Q-PCR (n=6). Data are expressed as transcript accumulation index (2–(ΔΔCT)) or TAI. c MDM were transfected with nanoplexes (24 h) then incubated in the presence of 10 μM methamphetamine (24 h). Gene expression for galectin-1 was examined using Q-PCR (n=4). Data are expressed as transcript accumulation index (2–(ΔΔCT)) or TAI. d Representative confocal image of galectin-1 protein expression (red) with DAPI nuclear staining (blue) following transfection of nanoplexes for 24 h then incubated in the presence of 10 μM methamphetamine for 24 h. a) scrambled control b) GNR c) control d) nanoplex e) methamphetamine f) nanoplex + methamphetamine. RFU are shown in each panel (n=4, scale bar=75.49 μM). All statistical significance was calculated using ANOVA followed by Bonferroni post-hoc test, * compared to control; † compared to methamphetamine alone. C = control; SCR = scramble; NP = nanoplex; ME = methamphetamine

Fig. 4

Concomitant galectin-1 and methamphetamine effects on HIV-1 infection. MDM were incubated with methamphetamine (10 μM, 24 h) alone or recombinant galectin-1 (2 μM, 30 min) alone or concomitantly prior to infection with HIV-1. a Supernatants were collected on day 5 and 10 post-infection as assayed for p24 antigen. Data are presented as p24 antigen levels (pg/ml) (n=6). b RNA was isolated on day 10 post-infection; HIV-LTR-R/U5 antigen gene expression was quantitated using Q-PCR (n=6). Data are expressed as transcript accumulation index (2–(ΔΔCT)) or TAI. All statistical significance was calculated using ANOVA followed by Bonferroni post-hoc test, * compared to control; † compared to methamphetamine alone; †† compared to recombinant galectin-1 alone

Fig. 5

Effect of nanoplex on HIV-1 infection. MDM were transfected with nanoplexes, 24 h post-transfection MDM were treated with methamphetamine (10 μM, 24 h) then infected with HIV-1 for 2 h, washed and returned to culture. a p24 antigen levels were measured at day, 5 and 10 post-infection (n=6). Data are expressed as pg/ml. b RNA was isolated on day 10 post-infection; HIV-LTR-R/U5 antigen gene expression was quantitated using Q-PCR (n=6). Data are expressed as transcript accumulation index (2–(ΔΔCT)) or TAI. All statistical significance was calculated using ANOVA followed by Bonferroni post-hoc test, * compared to control; † compared to methamphetamine alone; †† compared to recombinant galectin-1 alone