Preparation of nanoparticles and solutions

The nanoparticle powders silicon dioxide (10–20 nm, 637,238), aluminum oxide (~ 13 nm, 718,475) and iron(II, III) oxide (< 50 nm) (637,106) were purchased from Sigma-Aldrich (St. Louis, MO). For in vitro experiments, the PM samples were suspended in PBS and prepared in 10 mg/mL stock solutions. For in vivo experiments, the PM samples were suspended in saline to a stock solution concentration of 50 mg/mL. Then, all working solutions were mixed by ultrasonication for 15 min. The PMs suspensions were rolled for 10 s to ensure uniformity before each use and were then diluted to a working concentration of 30 μg/mL.

Influenza viruses

The seasonal influenza viruses H1N1 (A/New Caledonia/20/1999 (H1N1)) and avian influenza virus H5N1 (A/Jilin/9/2004 (H5N1)) were used in this study. Experiments with live influenza viruses were performed in biosafety level 3 facilities in accordance with governmental and institutional guidelines. Viruses were propagated and titered as described previously [17]. In brief, viruses were propagated by inoculation into 10- to 11-day-old, specific pathogen-free (SPF) embryonated chicken eggs and titered using the Reed-Muench method with MDCK cells. The titers are expressed as the median tissue culture infectious dose (TCID50) per milliliter of supernatant. Unless otherwise stated, a multiplicity of infection (M.O.I.) of 4 was used for H1N1 or H5N1 influenza virus infection in this study.

In vivo experiments

Wild-type C57BL/6 mice (6 to 8 weeks old; catalog no. 5,653,791, RRID MGI: 5,653,791) were purchased from Vital River (Beijing, China). Mice were first intratracheally injected with silica nanoparticles, and mice in the control group were administered allantoic fluid (AF). After 48 h, mice were intratracheally instilled with live H5N1 virus (106 TCID50). Mice were sacrificed 3 days after virus infection, and bilateral lungs were collected to assess lung injury and pulmonary edema. The animal experiments in this work were approved by the Ethics Committee of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (ACUC-A02-2017-014), and adhered to the Chinese National Guidelines for the Care and Use of Laboratory Animals and the institutional animal care guidelines.

Antibodies and reagents

Antibodies against LAMP1 (3243) (for immunoblotting) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against β-actin (clone AC-15, A5441) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Antibodies against LAMP2 (clone H4B4, sc-18,822) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-NP (C01321M) antibody was purchased from Millipore (Billerica, MA, USA), and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology. 2,2,2-Tribromoethanol (T48402) was purchased from Sigma-Aldrich.

Dulbecco’s modified essential medium (DMEM), fetal calf serum (FCS), and antibiotics were obtained from Gibco (Life Technologies) (Carlsbad, CA, USA). F-12/Ham’s nutrient medium and Halt Protease Inhibitor Cocktail were obtained from Thermo Fisher Scientific (Waltham, MA, USA), and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium inner salt (MTS) was procured from Promega Corporation (Madison, WI, USA). Acridine orange (AO) was acquired from Merck (Billerica, MA, USA). LysoTracker® Red DND-99 and ProLong® Gold Antifade Mountant with DAPI were purchased from Molecular Probes (Life Technologies) (Eugene, Oregon, USA).

Cell culture

The human lung adenocarcinoma A549 cell line was purchased from ATCC (Rockville, MA, USA) and cultured in F-12/Ham’s medium (HyClone) supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 units/mL penicillin, and 100 units/mL streptomycin at 37 °C in 5% CO2.

Immunoblot analysis

Immunoblot analysis was performed as described previously [18]. Cells were collected and lysed with RIPA lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate [SDS], 5 mM EDTA, 0.5% sodium deoxycholate, 1 mM Na3VO4, 1 mM NaF, and protease inhibitor cocktails). The total protein concentration of each sample was determined with a BCA protein assay kit (TianGen, Beijing, China), and samples were boiled with 2× loading buffer after equalization of the protein concentration. Samples were resolved by 10% or 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and proteins were transferred onto nitrocellulose filter membranes. Membranes were blocked with 2% albumin and were then incubated with the appropriate primary antibodies overnight prior to incubation with HRP-conjugated secondary antibodies at room temperature (RT) for 1 h. Binding of secondary antibodies was detected using a Kodak film exposure detection system, and the film was scanned and analyzed. For detection of a second primary antibody, nitrocellulose membranes were stripped with stripping buffer (1% SDS, 25 mM glycine [pH 2.0]) and were then incubated with another primary antibody.

Real-time quantitative PCR (q-PCR) analysis

Cells were lysed with TRIzol reagent (Invitrogen), and total RNA was isolated following the standard protocol provided by the manufacturer. Complementary DNA (cDNA) was synthesized from 1.5 μg of total RNA with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Life Technologies). PCR amplification was performed with FastStart Universal SYBR Green Master Mix and Rox (Roche) in a LightCycler 480 thermal cycler. The relative gene expression levels were calculated using the Ct values and were normalized to the expression levels of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the reference gene. The specific primers used were as follows: GAPDH forward: 5’-GGTGGTCTCCTCTGACTTCAACA-3’, GAPDH reverse: 5’-GTTGCTGTAGCCAAATTCGTTGT-3’; M1 forward: 5’- CTCTCTATCATCCCGTCAG-3’, M1 reverse: 5’-GTCTTGTCTTTAGCCATTCC-3’; M2 forward: 5’-ATTGTGGATTCTTGATCGTC-3’, M2 reverse: 5’-TGACAAAATGACCATCGTC-3’.

siRNA transfection

All siRNAs used in this study were designed and synthesized by RiboBio (Guangzhou, China). Before siRNA transfection, A549 cells were seeded at 3 × 105 cells/well in 12-well plates. Twenty-four hours later, cells were transfected with Lipofectamine RNAiMax reagent (Invitrogen) and the indicated siRNA (50 nM) diluted in Opti-MEM (Invitrogen) following the manufacturer’s guidelines. Forty-eight hours post siRNA transfection, downstream experiments were performed. The siRNA sequences for LAMP1 (siLAMP1) were 5’-CAAUGCGAGCUCCAAAGAAdTdT-3’/3’-dTdT GUUACGCUCGAGGUUUCUU-5’, and the siRNA sequences for LAMP2 (siLAMP2) were 5’- GCGGUCUUAUGCAUUGGAAdTdT-3’/3’-dTdT CGCCAGAAUACGUAACCUU-5’.

Confocal microscopy

A549 cells were grown on coverslips in 24-well plates. At the indicated time points after virus infection, the culture supernatant was removed, and the coverslips were washed three times with PBS. Then, the cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked with 10% goat serum for an additional 30 min. The coverslips were then incubated first with the indicated primary antibody at RT for 1 h and then with an Alexa Fluor 488- or Alexa Fluor 568-labeled secondary antibody (Molecular Probes). The subcellular localization of each target protein was observed using confocal laser scanning microscopy (FV-1000, Olympus, Tokyo, Japan), and the images were analyzed with Olympus Fluoview 3.0 software. For analysis of NP-positive nuclei, at least 64 images were continuously acquired automatically by the confocal microscope and were assessed with ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).

To label cellular lysosomes, cells were incubated with 300 nM LysoTracker Red DND-99 (Molecular Probes) in fresh medium for 30 min at 37 °C and were then extensively washed with PBS and fixed with 4% paraformaldehyde for 15 min. Finally, the coverslips were mounted with ProLong® Gold Antifade Mountant with DAPI.

Cell viability assays

A549 cells were infected with virus at an M.O.I. of 4 or with an equal volume of vehicle for the indicated durations. Cell viability was then determined by an MTS assay (Promega, Madison, WI, USA). In the treatment experiments, nanoparticles (30 μg/mL) were added 1 h before virus administration or at the indicated time points. The MTS assay was performed 48 h post virus infection.

Nanoparticle and influenza virus instillation, lung wet/dry ratio assay and histopathological examination of mouse lung tissue

Mice were randomly grouped. After anesthesia was induced by an intraperitoneal injection of 2,2,2-tribromoethanol (500 mg/kg body weight), the tracheas of the mice were separated surgically. The nanoparticle suspensions were administered by intratracheal instillation (30 mg/kg body weight). Instillation was selected as the exposure method over other methods, such as inhalation and aspiration, because of the comparative accuracy of the nanoparticle intake during manipulation. Saline was used as the control. After 48 h, a second tracheal separation under anesthesia was performed, and H5N1 and H1N1 viruses (TCID50 = 106, 4 μL/g body weight) were intratracheally instilled. AF was used as the control. Seventy-two hours post -influenza virus infection, mice were sacrificed. The lung tissues were rapidly collected for further examination. In the lung wet/dry ratio assay, which allows evaluation of pulmonary edema, the wet weight of the lungs was measured. To obtain the dry weight, the lungs were dried at 65 °C for 24 h. For histopathological examination, lungs were fixed in formalin for 48 h and embedded in paraffin, and 4-μm sections were stained with hematoxylin and eosin (H&E). Micrographs were acquired with a Leica DM3000 microscope connected to a Leica DFC 420 digital camera and Leica Qwin V3 software. The number of neutrophils per field of view (400×) was determined in 100 microscopic fields per group.

Statistical analysis

All data are shown as the mean ± S.E.M. values. Measurements at single time points were analyzed by ANOVA and, if the differences were significant, the measurements were further analyzed by a two-tailed t-test. All statistical tests were conducted using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). P < 0.05 indicates a statistically significant difference; P < 0.01, a highly significant difference (**).