Nanodiscs as a therapeutic delivery agent: inhibition of respiratory syncytial virus infection in the lung

Abstract

There is increasing interest in the application of nanotechnology to solve the difficult problem of therapeutic administration of pharmaceuticals. Nanodiscs, composed of a stable discoidal lipid bilayer encircled by an amphipathic membrane scaffold protein that is an engineered variant of the human Apo A-I constituent of high-density lipoproteins, have been a successful platform for providing a controlled lipid composition in particles that are especially useful for investigating membrane protein structure and function. In this communication, we demonstrate that nanodiscs are effective in suppressing respiratory syncytial viral (RSV) infection both in vitro and in vivo when self-assembled with the minor pulmonary surfactant phospholipid palmitoyloleoylphosphatidylglycerol (POPG). Preparations of nanodiscs containing POPG (nPOPG) antagonized interleukin-8 production from Beas2B epithelial cells challenged by RSV infection, with an IC50 of 19.3 μg/mL. In quantitative in vitro plaque assays, nPOPG reduced RSV infection by 93%. In vivo, nPOPG suppressed inflammatory cell infiltration into the lung, as well as IFN-γ production in response to RSV challenge. nPOPG also completely suppressed the histopathological changes in lung tissue elicited by RSV and reduced the amount of virus recovered from lung tissue by 96%. The turnover rate of nPOPG was estimated to have a halftime of 60-120 minutes (m), based upon quantification of the recovery of the human Apo A-I constituent. From these data, we conclude that nPOPG is a potent antagonist of RSV infection and its inflammatory sequelae both in vitro and in vivo.

Keywords: anti-viral; innate immunity; nanodiscs; phospholipids; therapeutic delivery.

Figure 1

Molecular dynamic simulated model of Nanodisc. Notes: Nanodiscs consist of a phospholipid bilayer encircled by two molecules of an Apo-A1-derived membrane scaffold protein, which organizes the lipids and shields the acyl chains from the aqueous environment. Abbreviation: MSP, membrane scaffold protein.

Figure 2

nPOPG inhibits RSV-induced IL-8 production by Beas2B cells. (A) IL-8 production by the Beas2B cells was measured by ELISA after either sham treatment or virus infection for 48 hours, in either the absence or presence of 200 μg/mL POPG, POPC added in the form of liposomes (+POPG), or nanodiscs (+nPOPG, +nPOPC). The cells were infected at a multiplicity of infection (0.5). Values shown are means ± SE for three independent experiments. *indicates P < 0.001. The average production of IL-8 with RSV infection alone in the three experiments was 3977.6 ± 865 pg/mL (B) Flagellin (10 ng/mL)-induced IL-8 production by the Beas2B cells was measured by ELISA after 48 hours of incubation. The flagellin challenge was performed in either the absence or presence of 200 μg/mL of nPOPG, nPOPC, or liposomes (+POPG), as indicated. The treatment of the cells with nPOPG or nPOPC in the absence of flagellin challenge is also shown in the figure. Note: The data shown are from three independent experiments. Abbreviations: CONL, uninfected control; ELISA, enzyme linked immunosorbent assay; Fla, flagellin; POPG, liposomal palmitoyl-oleoyl-phosphatidylglycerol; nPOPG, nanodisc palmitoyl-oleoyl-phosphatidylglycerol; POPC, liposomal palmitoyl-oleoyl-phosphatidylcholine; nPOPC, nanodisc palmitoyl-oleoyl-phosphatidylcholine; RSV, respiratory syncytial virus; SE, standard errors of the mean.

Figure 3

Nanodisc POPG suppresses RSV-elicited IL-8 production in a concentration- and phospholipid class-dependent manner and inhibits the cytopathic effects of RSV upon Beas2B cells. (A) IL-8 production by Beas2B cells was measured by ELISA after challenging cultures with RSV (multiplicity of infection = 0.5) for 48 hours in either the absence or presence of varying concentrations of liposome POPG, nPOPG, or nPOPC. At the end of the incubation period, the culture medium was harvested and analyzed for the presence of secreted IL-8. Data shown are the means ± SE for three experiments. (B) Beas2B cells were either sham-treated or infected with a virus (RSV) for 48 hours, in either the absence or presence of 200 μg/mL nPOPG or nPOPC, as indicated. The cell monolayers were visualized by phase contrast microscopy. Note: The bar inset in the CONL panel corresponds to 50 μm. Abbreviations: CONL, uninfected control; POPG, palmitoyl-oleoyl-phosphatidylglycerol; nanoPOPG, nanodisc palmitoyl-oleoyl-phosphatidylglycerol; nanoPOPC, nanodisc palmitoyl-oleoyl-phosphatidylcholinel; RSV, respiratory syncytial virus; SE, standard errors of the mean.

Figure 4

nPOPG inhibits RSV infection of HEp2 cells in tissue culture. Notes: Monolayers of HEp2 cells were incubated in six well plates for 1 h prior to the addition of RSV either without lipids or with 200 μg of liposome POPG or nPOPG. Serial 10-fold dilutions of RSV from 2 × 10⁴ − 2 × 10² infectious particles per mL were added to duplicate wells in the absence or presence of lipids. After a viral adsorption period of 1 h, the unbound virus and the lipid were removed from each well, and the monolayers were overlaid with an agar-containing medium for the development of plaques. After 6 d, the cultures were fixed with 1% buffered formalin and stained with 0.05% neutral red, and plaques were counted. The data shown are means ± SE from three independent experiments. *indicates P < 0.001. Abbreviations: POPG, liposomal palmitoyl-oleoyl-phosphatidylglycerol; nPOPG, nanodisc palmitoyl-oleoyl-phosphatidylglycerol; RSV, respiratory syncytial virus; SE, standard errors of the mean.

Figure 5

nPOPG inhibits the production of RSV mRNA. (A) Monolayers of HEp2 cells were pretreated with 200 μg/mL nPOPG or nPOPC for 1 h prior to the addition of RSV (multiplicity of infection = 1 × 10⁻³). After 2 h of RSV adsorption in the absence or presence of lipids, the cells were washed three times with PBS and then grown in DMEM/F12, either with or without 200 μg/mL nPOPG or nPOPC. The complete contents of each well were processed for RNA extraction at various time points (from 2 h to 96 h) and subjected to analysis by quantitative RT-PCR and gel electrophoresis. (B) The results of two independent experiments are shown. Note: Values are means ± SD. Abbreviations: MOI, multiplicity of infection; nPG, nanodisc palmitoyl-oleoyl-phosphatidylglycerol; nPC, nanodisc palmitoyl-oleoyl-phosphatidylcholine; F-mRNA, messenger RNA encoding viral F-glycoprotein; RSV, respiratory syncytial virus.

Figure 6

nPOPG blocks RSV binding to epithelial cells. (A) Monolayers of HEp2 cells, at 18°C, were challenged with RSV at MOI = 0–10, as indicated, in either the absence or presence of 200 μg/mL nPOPC, nPOPG, or liposome POPG. UN identifies cultures that were not infected with the virus. The cultures were washed with PBS three times and harvested and processed for SDS-PAGE and immunoblotting. GP indicates the location of the viral G protein, and β-actin blotting was used to normalize the recovery of cells from each well. (B) The quantification of three independent immunoblotting experiments is shown. Note: Values are means ± SE for three independent experiments. Abbreviations: MOI, multiplicity of infection; nanoPG, nanodisc palmitoyl-oleoyl-phosphatidylglycerol; nanoPC, nanodisc palmitoyl-oleoyl-phosphatidylcholine; UN, unifected control; GP, viral G-glycoprotein; PFU, plaque forming units; RSV, respiratory syncytial virus; SE, standard errors of the mean.

Figure 7

nPOPG inhibits RSV infection and inflammation in vivo. BALB/c mice were infected with RSV (1 × 10⁷ pfu/mouse) by intranasal inoculation in either the absence or presence of liposome POPG or nPOPG (400 μg phospholipid), as indicated. The mice were euthanized 5 days after infection and their lungs were lavaged and subsequently removed for performing histopathology and quantitative plaque assays. The lavage material was used for analysis of cell populations and quantification of IFN-γ. (Panel A) shows the total cell numbers in BALF. (Panel B) shows the inflammatory cell populations in BALF. (Panel C) shows the IFN-γ levels in BALF as determined by ELISA. (Panel D) shows RSV titer in tissue homogenates prepared from the left lung in each group as measured by quantitative plaque assay. Notes: Values shown are the means ± SE for two independent experiments: *P < 0.001, ^§P < 5 × 10⁻⁵. In each experiment, every group contained five to eight mice. Abbreviations: CONL, uninfected control; nPOPG, nanodisc palmitoyl-oleoyl-phosphatidylglcyerol; POPG, liposomal palmitoyl-oleoyl-phosphatidylglycerol; RSV, respiratory syncytial virus; SE, standard errors of the mean.

Figure 8

nPOPG suppresses lung inflammation elicited by RSV. (A) Lungs from sham-infected (CONL) or RSV-infected mice treated with nPOPG were fixed in 10% buffered formalin, embedded in paraffin, and stained with hematoxylineosin. Lung sections from each animal were evaluated and given a histopathology score. Values shown are the means ± SE. *P = 4.1 × 10⁻⁶. (B) Representative micrographs from the CONL- and virus-infected groups treated with 400 μg/mL nPOPG. Note: The bar inset in the CONL panels corresponds to 200 μm. Abbreviations: CONL, uninfected control; nPOPG, nanodisc palmitoyl-oleoyl-phosphatidylglycerol; RSV, respiratory syncytial virus; SE, standard errors of the mean.

Figure 9

In vivo turnover of nPOPG in the lung. (A) Mice were anesthetized and inoculated with 400 μg nPOPG and euthanized at the indicated times. The euthanized animals were lavaged with one mL of PBS and the cell-free recovered material was subjected to electrophoresis and quantitative immunoblotting using anti-human Apo A-I antibodies. Samples from all time points were diluted 20-fold from the original BALF sample. (B) Images in (A) were quantified using NIH-Image J software. Note: Values are shown as the means ± SE for three independent experiments, with four to eight mice/time point in each experiment. Abbreviations: KD, kilo Dalton; nano POPG, nanodisc palmitoyl-oleoyl-phosphatidylglycerol; RSV, respiratory syncytial virus; SE, standard errors of the mean.