Nanoparticle synthesis
NGs, crosslinked protein nanoparticles, charged protein nanoparticles, liposomes,and polystyrene nanoparticles were prepared as previously described36,43. Details of the synthesis for each type of nanoparticle are included in the Supplementary Information. Nanoparticles, proteins and bacteria were labelled with 125I, 131I or 111In according to described methods57. Details of labelling techniques are provided in the Supplementary Information.
E. coli preparation
TOP10 E. coli (ThermoFisher #C404002) were grown overnight in Terrific broth with ampicillin. Bacteria were heat-inactivated by a 20 min incubation at 60 °C, then fixed by overnight incubation in 4% paraformaldehyde. After fixation, bacteria were pelleted by centrifugation at 1,000g for 10 min. Pelleted bacteria were washed three times in PBS, before resuspension by pipetting. Bacterial concentration was verified by optical density at 600 nm, before radiolabelling as described above. Bacteria were administered i.v. in mice (7.5 × 107 colony-forming units in a 100 µl suspension per mouse).
Nanoparticle and protein tracing in mice
Nanoparticle or protein biodistributions were tested by i.v. injecting nanoparticles or protein (suspended to 100 µl in PBS or 0.9% saline at a dose of 2.5 mg kg−1 with tracer quantities of radiolabelled material) in C57BL/6 male mice from Jackson Laboratories. For experiments tracing anti-Ly6G biodistributions to locate intravascular neutrophils, radiolabelled anti-Ly6G was administered i.v. at 0.1 mg kg−1. The quantity of injected radioactivity was measured by gamma counter (Perkin-Elmer) immediately before injection. To determine the nanoparticle masses for dosing, NGs and charged protein nanoparticles were prepared from reactants at known concentrations in synthetic methods involving no material loss; crosslinked protein nanoparticles were resuspended from weighed powder; polystyrene nanoparticles, ferritin and viral capsids were purchased at known concentrations. Liposomes were prepared from reactants at known concentrations and previous work with the same synthetic methods assessed low material losses during filtration and purification of liposomes57,71.
Biodistributions in naive mice were compared to biodistributions in injury models. Biodistribution data were collected at 30 min after nanoparticle or protein injection, unless otherwise stated. Blood was collected by vena cava draw and mice were killed by exsanguination and cervical dislocation. Organs were harvested and rinsed in saline, and blood and organs were examined for nanoparticle or protein retention by gamma counter. To calculate concentration in organs, quantity of retained radioactivity was normalized to organ weights.
For i.v. LPS, mice were anaesthetized with 3% isoflurane before retro-orbital injection of LPS from E. coli strain B4 at 2 mg kg−1 in 100 µl PBS. After 5 h, mice were anaesthetized with ketamine/xylazine (10 mg kg−1 ketamine, 100 mg kg−1 xylazine, intramuscular administration) and before jugular vein nanoparticle or protein injection. For i.t. LPS, B4 LPS was administered to mice (anaesthetized with ketamine/xylazine) at 1 mg kg−1 in 50 µl of PBS via tracheal catheter, followed by 100 µl of air71. NGs were injected 16 h after i.t. LPS and DBCO–IgG liposomes were injected 1, 2 or 6 h after i.t. LPS. For footpad LPS, B4 LPS was administered at 1 mg kg−1 in 50 µl PBS via footpad injection. NGs were injected i.v. 6 or 24 h after footpad LPS. For cardiogenic pulmonary oedema, mice were anaesthetized with ketamine/xylazine and administered propranolol in saline (3 µg ml−1) via jugular vein catheter at 83 µL min−1 over 120 min62, before i.v. NG injection. For localized footpad inflammation, mice were anaesthetized with 3% isoflurane and 20 µl of CFA or 20 µl of sham saline was injected subcutaneously in the central plantar region of the left hind paw42, 6 h before i.v. NG injection.
Single-cell suspension flow cytometry
Single-cell suspensions were prepared from male C57BL/6 mouse lungs for flow cytometry. Fluorescent nanoparticles were administered at 2.5 mg kg−1 30 min before the animals were killed and their lungs extracted. Mice were anaesthetized with ketamine/xylazine (10 mg kg−1 ketamine, 100 mg kg−1 xylazine, intramuscular administration) before installation of a tracheal catheter secured by suture. After the animals had been killed by vena cava exsanguination, lungs were perfused via the right ventricle with ~10 ml of cold PBS. Lungs were then infused via a tracheal catheter with 1 ml of cold PBS solution with 5 U ml−1 dispase, 2.5 mg ml−1 collagenase I and 1 mg ml−1 of DNAse I. Immediately after infusion, the trachea was sutured shut while removing the tracheal catheter. Lungs with intact trachea were removed via thoracotomy and kept on ice before manual disaggregation.
Single-cell suspensions were also prepared from mouse feet. Feet were removed immediately following after the animals had been killed by cervical dislocation and 100 µl of dispase/collagenase/DNAse was injected subcutaneously in the feet. Tissue was separated from the bones while the feet were held in 1 ml of dispase/collagenase/DNAse.
Disaggregated lung or foot tissue was aspirated in an additional 2 ml of dispase/collagenase/DNAse and incubated at 37 °C for 45 min, vortexing every 10 min. After addition of 1 ml of fetal calf serum, tissue suspensions were strained through 100 µm filters and centrifuged at 400g for 5 min. Pelleted material was resuspended in 10 ml of cold ACK lysing buffer. The resulting suspensions were strained through a 40 µm filter, incubated for 10 min on ice, and centrifuged at 400g for 5 min. The pelleted material was rinsed in 10 ml of FACS buffer (2% fetal calf serum and 1 mM EDTA in PBS). After 400g/5 min centrifugation, pellets were resuspended in 2% PFA in 1 ml FACS buffer for a 10 min room temperature incubation. Fixed cell suspensions were centrifuged at 400g for 5 min and resuspended in 1 ml of FACS buffer.
To stain fixed cells, 100 µl aliquots of cell suspensions were pelleted at 400g for 5 min, then resuspended in labelled antibody diluted in FACS buffer (1:150 dilution for anti-Ly6G, APC-anti-CD31 or PE-anti-F480 antibodies and 1:500 dilution for anti-CD45 andtibodies). Samples were incubated with staining antibodies for 20 min at room temperature in the dark, diluted with 1 ml of FACS buffer and pelleted at 400g for 5 min. Stained pellets were resuspended in 200 µl of FACS buffer immediately before flow cytometry (BD Accuri). Data were gated to exclude debris and doublets. Controls with no stain, obtained from naive and i.v.-LPS-injured mice, established gates for negative/positive staining with FITC/AlexFluor 488, PE, PerCP/Cy5.5 and APC/Alexa Fluor 647. Single-stain controls allowed automatic generation of compensation matrices in FCS Express software.
To analyse intravascular leukocyte populations in lungs, mice received i.v. FITC-anti-CD45 5 min before they were killed. Populations of intravascular versus extravascular leukocytes were assessed by also staining fixed cell suspensions with PerCP-conjugated anti-CD45 and/or Alexa Fluor 647-conjugated anti-Ly6G. Comparing PerCP anti-CD45 with FITC anti-CD45 signal indicated intravascular versus extravascular leukocytes. Comparison of Alexa Fluor 647 anti-Ly6G, PerCP anti-CD45 and FITC anti-CD45 signal indicated intravascular versus extravascular neutrophils.
To characterize nanoparticle distribution among different cells in lungs or feet, fluorescent nanoparticles were administered at 2.5 mg kg−1 via jugular vein injection and circulated for 30 min. Fixed single-cell suspensions were stained and coincidence of nanoparticle fluorescence with anti-CD45, anti-Ly6G, PE anti-CD31 or PE anti-F480 fluorescence was assessed.
In vitro neutrophil uptake of nanoparticles
Bone marrow was collected from pooled femurs of C57BL/6 mice. Neutrophils were isolated with the StemCell Technologies RoboSep Mouse Neutrophil Enrichment Kit by magnetic-bead-mediated depletion of non-neutrophils. To serum-treat NGs before incubation with neutrophils, 5 × 109 FITC-labelled NGs in 10 µl PBS were incubated with 10 µl serum for 1 h at 37 °C; then 1 × 106 neutrophils were rotated with 5 × 109 NGs in 20 µl PBS for 15 min at 37 °C. For flow cytometry (BD Accuri C6), neutrophils were washed and stained with PerCP/Cy5.5 Ly6G antibodies (BD Biosciences, 1:100 dilution) and non-neutrophils were excluded from analysis via Ly6G staining (see Supplementary Fig. 27 for gating). NG fluorescence in neutrophils was quantified.
To probe the role of complement in neutrophil–nanoparticle interactions, complement was depleted from serum via two methods: heat treatment and CVF treatment. For heat treatment, serum was incubated at 56 °C for 1 h and denatured proteins were removed by centrifugation at 10,000g for 15 min. For CVF treatment, 10 units CVF per ml serum were incubated for 1 h at 37 °C, then centrifuged at 10,000g for 15 min. Nanoparticle incubation with heat- and CVF-treated serum followed the same protocol described above for naive serum.
In vivo effects of complement on nanoparticle tropism for neutrophils
Mice were dosed with 100 µg CVF per kg via intraperitoneal injection 24 h before NG administration or blood draw to test the in vivo effects of complement depletion on nanoparticle–neutrophil interactions. For experiments with both LPS and CVF, B4 LPS from E. coli was administered i.v. at 2 mg kg−1 19 h after CVF and 5 h before i.v. NGs (2.5 mg kg−1), as described in Nanoparticle and protein tracing in mice.
Mass spectrometry profiles of protein coronae on nanoparticles
A 25 µl volume of NGs or adenovirus capsids in a 5 mg ml−1 suspension were incubated with and equivalent volume of wild-type or CVF-treated (as above) mouse serum or saline sham for 1 h at 37 °C. Nanoparticles were pelleted by centrifugation and washed with 1 ml PBS three times to separate from unbound serum proteins.
Opsonized and sham-opsonized nanoparticles were prepared for mass spectrometry analysis as follows. Samples were solubilized and digested with the iST kit (PreOmics GmbH) per the manufacturer’s protocol. Nanoparticle pellets were resuspended, reduced and alykylated by addition of sodium deoxycholate buffer containing tris(2-carboxyethyl)phosphine and 2-chloroacetamide. The resulting suspensions were heated at 95 °C for 10 min. Proteins were enzymatically hydrolysed for 1.5 h at 37 °C by endoproteinase Lys-C and trypsin. The resulting peptides were desalted, dried by vacuum centrifugation and reconstituted in 0.1% trifluoroacetic acid containing indexed retention time peptides (Biognosys Schlieren). Ultraperformance liquid chromatography–mass spectrometry data were obtained and analysed by published methods72,73, as detailed in the Supplementary Information.
SPECT/CT imaging
Imaging techniques, as described previously57, are detailed in the Supplementary Information. SPECT and CT data, in NIFTI format, were opened with ImageJ software (FIJI package) and processed for background removal, pseudocolour assignment and three-dimensional reconstruction, as detailed in the Supplementary Information.
Nanoparticle administration in human lungs
Human lungs were obtained after organ harvest from transplant donors whose lungs were in advance deemed unsuitable for transplantation. Lungs were kept at 4 °C and used within 24 h of organ harvest. Lungs were inflated with low-pressure oxygen and oxygen flow was maintained at 0.8 l min−1 for gentle inflation. Pulmonary artery subsegmental branches were endovascularly cannulated, then tested for retrograde flow by perfusing for 5 min with Steen solution containing a small amount of green tissue dye at 25 cm H2O pressure. Pulmonary veins through which efflux of perfusate emerged were noted, allowing collection of solutions after passage through the lungs. A 2 ml mixture of 125I-labelled NGs and 131I-labelled ferritin was injected through the arterial catheter. Approximately 100 ml of 3% BSA in PBS was passed through the same catheter to rinse unbound nanoparticles. A solution of green tissue dye was subsequently injected through the same catheter. The cannulated lung lobe was dissected into ~1 g segments, which were evaluated for density of tissue dye staining. Segments were weighed, divided into ‘high’, ‘medium’, ‘low’ and ‘null’ levels of dye staining, and measured for 131I and 125I signal in a gamma counter.
For experiments with cell suspensions derived from human lungs (chosen for research use as above), single-cell suspensions were generously provided by Edward Morrisey at the University of Pennsylvania. Aliquots of 600,000 cells were pelleted at 400g for 5 min and resuspended in 100 µl FACs buffer containing different quantities of FITC-dextran NGs. Cells and NGs were incubated at room temperature for 60 min before twofold pelleting at 400g with 1 ml PBS washes. Cells were resuspended in 200 µl FACS buffer for staining with APC anti-human CD45 (1:500 dilution, 20 min room temperature incubation). Cells were pelleted at 400g for 5 min and resuspended in 200 µl PBS for immediate analysis with flow cytometry (BD Accuri). Negative/positive NG or anti-CD45 signal was established by comparison to unstained cells. Single-stained controls indicated no spectral overlap between FITC-NG fluorescence and anti-CD45 APC fluorescence.
Effects of nanoparticles in nebulized LPS model
Mice were exposed to nebulized B4 LPS in a whole-body exposure chamber, with separate compartments for each mouse (MPC-3 AERO; Braintree Scientific). To maintain adequate hydration, mice were injected with 1 ml sterile saline warmed to 37 °C, intraperitoneally, immediately before LPS exposure. LPS was reconstituted in PBS to 10 mg ml−1 and stored at −80 °C until use. Immediately before nebulization, LPS was thawed and diluted to 5 mg ml−1 with PBS. Then, 5 ml of diluted LPS was aerosolized via a jet nebulizer connected to the exposure chamber (NEB-MED H, Braintree Scientific). Nebulization was performed until all liquid was nebulized (~20 min).
DBCO–IgG liposomes (20:1 DBCO:IgG, 2.5, 5,10 or 30 mg kg−1), bare liposomes (30 mg kg−1), NGs (30 mg kg−1) or saline sham were administered via retro-orbital injections of 100 µl of suspension 2 h after LPS exposure. Mice were anaesthetized with 3% isoflurane to facilitate injections. Blood draws and BALF were collected 24 h after LPS exposure, as previously described and detailed in the Supplementary Information71. Mice were weighed before administration of nebulized LPS and before BALF and blood draws.
To stain for flow cytometry, BALF samples were centrifuged at 300g for 4 min, the supernatant was aspirated and 100 µl of staining buffer (1:1,000 APC-anti-CD45 or 1:150 Alexa Fluor 488-anti-Ly6G in FACS buffer) was added. Samples were stained for 30 min at room temperature in the dark, then 1 ml of FACS buffer was added, samples were centrifuged at 300g for 4 min and supernatant was aspirated. Cells were resuspended in 900 µl of FACS buffer for flow cytometry analysis (BD Accuri). Forward scatter (area) versus side scatter (area) plots gated-out non-cellular debris and forward scatter (area) versus forward scatter (height) plots gated-out doublets. Unstained controls set gates for APC and Alexa Fluor 488 signal. Single-stained controls showed no spectral overlap between APC-anti-CD45 and Alexa Fluor 488-anti-Ly6G. CD45- and Ly6G-positive cells determined leukocyte and neutrophil concentrations, respectively.
To trace intravascular neutrophils after nebulized LPS treatment and 10 mg kg−1 DBCO–IgG liposome dosing, 125I-anti-Ly6G (0.1 mg kg−1) was administered 1 or 22 h after liposomes, and biodistributions were determined as described above. In mice treated with DBCO–IgG liposomes, blood was drawn into EDTA via the vena cava before exsanguination. Lungs and liver were removed after obtaining BALF and stored at −80 °C. Blood was immediately evaluated with CBC measurements (Abaxis VetScan HM5). Blood remaining after CBC was centrifuged at 1,500g for 10 min at 4 °C and plasma was extracted and stored at −80 °C. Chemokine CXCL2 and cytokine IL-6 were measured in BALF, plasma, and lung and liver homogenates according to published methods detailed in the Supplementary Information74.
CD spectroscopy
Proteins were prepared in deionized and filtered water at concentrations of 0.155 mg ml−1 for human albumin, 0.2 mg ml−1 for hen lysozyme and 0.48 mg ml−1 for IgG. Albumin NPs, NGs and IgG-coated liposomes were diluted such that albumin, lysozyme and IgG concentrations in the suspensions matched concentrations of corresponding protein solutions. Protein and nanoparticle solutions were analysed in quartz cuvettes with 10 mm path length in an Aviv CD spectrometer. The instrument was equilibrated in nitrogen at 25 °C for 30 min before use and samples were analysed with sweeps between 185 and 285 nm in 1 nm increments. Each data point was obtained after a 0.333 s settling time, with a 2 s averaging time. CDNN75 software deconvoluted CD data (expressed in millidegrees) via a neural network algorithm assessing alignment of spectra with library-determined spectra for helices, antiparallel sheets, parallel sheets, β turns and random coils75.
8-Anilino-1-naphthalenesulfonic acid nanoparticle staining
8-Anilino-1-naphthalenesulfonic acid (ANSA) at 0.06 mg ml−1 was mixed with lysozyme, human albumin or IgG at 1.5 mg ml−1 in PBS. For nanoparticle analysis, nanoparticle suspensions were prepared such that albumin, lysozyme and IgG concentrations in the suspensions matched the 1.5 mg ml−1 concentration of protein solutions. Protein or nanoparticles and ANSA were reacted at room temperature for 30 min. Excess ANSA was removed from solutions by three centrifugations against 3 kDa cut-off centrifugal filters (Amicon). After resuspension to original volume, ANSA-stained protein/nanoparticle solutions/suspensions were examined for fluorescence (excitation, 375 nm) and absorbance maxima corresponding to ANSA.
Histology
For imaging neutrophils in naive and i.v.-LPS-affected lungs, mice were given i.v. anti-Ly6G and killed 30 min later. Lungs were embedded in M1 medium, flash frozen and sectioned in 10 µm slices. Sections were stained with Alexa Fluor 594-goat anti-rat secondary antibody (1:200 dilution) and imaged with epifluorescence microscopy. Similarly, rhodamine–dextran NGs were administered i.v. in i.v.-LPS mice 30 min before the mice were killed. Lungs were sectioned as above and stained with clone 1A8 anti-Ly6G antibody, followed by Alexa Fluor 350-goat anti-rat secondary antibody (1:150 dilution), before epifluorescence and confocal imaging of NG and neutrophil fluorescence.
For histological verification of injury following nebulized LPS treatment, one set of injured mouse lungs and one set of naive lungs was infused intratracheally with 4% paraformaldehyde. The trachea was tied off and lungs and trachea were removed via thoracotomy. The lungs were suspended in 4% paraformaldehyde for overnight fixation before embedding in paraffin, sectioning and haematoxylin/eosin staining.
Sections of human lungs were obtained after administration of rhodamine–dextran NGs. NG-perfused and non-perfused tissue regions were harvested, embedded in M1 medium, flash frozen and sectioned in 10 µm slices. NG fluorescence and tissue autofluorescence were detected with epifluorescence imaging.
Live lung imaging
A mouse was anaesthetized with ketamine/xylazine 5 h after i.v. LPS. A jugular vein catheter was placed for injection of NGs, anti-CD45 and fluorescent dextran. A patch of skin on the back of the mouse, around the juncture between the ribcage and the diaphragm, was denuded. The mouse was maintained on mechanical ventilation and the lungs were exposed via incision at the juncture between the ribs and the diaphragm. A coverslip affixed to a rubber O-ring was sealed to the incision by vacuum. The exposed lungs were focused under the objective using autofluorescence. With 100 ms exposure, channels corresponding to violet, green, near-red and far-red fluorescence were sequentially imaged. Rhodamine–dextran NGs (2.5 mg kg−1), Brilliant Violet-anti-CD45 (0.8 mg kg−1) and Alexa Fluor 647–70 kDa dextran (40 mg kg−1) were injected via the jugular vein. Images were recorded for 30 min in SlideBook software and opened in ImageJ (FIJI distribution) for composition in movies with co-registration of the four fluorescent channels.
Animal and human study protocols
All animal studies were carried out in strict accordance with Guide for the Care and Use of Laboratory Animals as adopted by National Institutes of Health and approved by University of Pennsylvania Institutional Animal Care and Use Committee. All animal experiments used male C57BL/6 J mice, 6–8 weeks old, purchased from Jackson Laboratories. Mice were maintained at 20–25 °C, 50% ± 20% humidity, and on a 12/12 h dark/light cycle with food and water ad libitum.
Human lungs were obtained by the University of Pennsylvania Lung Biology Institute’s Human Lung Tissue Bank (HLTB) from Gift of Life Donor Program (Philadelphia, PA, USA). Lungs provided to the HLTB were determined unsuitable for transplantation into a recipient, and would have been discarded if not used for our studies. Lungs provided by the HLTB for these studies are deidentified and cannot be linked to individual donors. Clinical staff procuring and distributing the tissue through the HLTB are not involved in the research after distribution of the deidentified tissue. Studies employing deidentified tissue from the HLTB were therefore determined by the University of Pennsylvania Institutional Review Board (IRB) to be IRB-exempt and were not considered human research subjects as defined by the Office of Human Research Protection of the National Institutes of Health. Deidentified patient data for human lungs (age, sex and cause of death) are tabulated in Supplementary Table 11.
Statistical analysis
Error bars indicate the standard error of the mean throughout. Significance tests are described in captions. Statistical power was determined for statements of statistical significance and tabulated in the supplementary materials.
Reporting Summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
