Supporting Information

Static and dynamic microscopy of the chemical stability and aggregation state of silver

nanowires in components of murine pulmonary surfactant

Ioannis G. Theodoroua, Danielle Botelhob, Stephan Schwanderc, Junfeng (Jim) Zhangd, Kian

Fan Chunge, Teresa D. Tetleye, Milo S. P. Shafferf, Andrew Gowb, Mary P. Ryan*,a and

Alexandra E. Porter*,a

aDepartment of Materials and London Centre for Nanotechnology, Imperial College London,

Exhibition Road, London SW7 2AZ, United Kingdom

bDepartment of Pharmacology and Toxicology, Rutgers University, Piscataway, New Jersey

08854, United States

cRutgers School of Public Health, Department of Environmental and Occupational Health,

Piscataway, New Jersey 08854, United States

dNicholas School of the Environment and Duke Global Health Institute, Duke University,

Durham, NC 27708, United States

eNational Heart and Lung Institute, Imperial College London, London SW3 6LY, United

Kingdom

fDepartment of Chemistry and London Centre for Nanotechnology, Imperial College London,

Exhibition Road, London SW7 2AZ, United Kingdom

Corresponding Authors:

*(A.E.P.): a.porter@imperial.ac.uk

*(M.P.R.): m.p.ryan@imperial.ac.uk

Number of pages: 14

Number of figures: 3

Number of tables: 0

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Methods

AgNW Synthesis:

Pure AgNWs were synthesized using a modified polyol process, originally developed

by Xia et al.1 Ethylene glycol (EG) acts as both solvent and reducing agent, whereas

poly(vinyl pyrrolidone) (PVP) is used as the capping agent. The reduction of Ag+ ions by EG

leads to the formation of Ag nuclei at the early stages of the reaction. Due to the stronger

affinity of PVP for the (100) facets than the (111) facets of the Ag nuclei, this passivation

leads to one-dimensional growth and the formation of high aspect-ratio AgNWs. The process

was optimized in order to: (i) Eliminate the generation of AgNPs from the synthesis product,

as the presence of different populations of particles would confound the correlation between

the observed effects and the physicochemical properties of the particles. (ii) Avoid the use of

other transition metal impurities, such as Cu 2 or Fe,3 previously used to control the product

morphology in the polyol synthesis of Ag nanostructures, because their impact on the

physicochemical properties of AgNWs is not well-understood. (iii) Eliminate the need to

control the rate of injection of the reactants, making the synthesis less complicated.4

Briefly, Ethylene Glycol (EG, Sigma-Aldrich, anhydrous, 99.8%) (2.5 mL) was

placed in a double-neck round-bottom flask connected to a condenser. A stock solution of

sodium chloride (NaCl) (0.05 M) was prepared by dissolving NaCl in EG by bath sonication.

The appropriate amount of NaCl stock solution was added to the flask so that the

concentration of NaCl in the final reaction volume was 60 μM. The flask was heated in an oil

bath at 160 oC for 30 minutes to remove trace amounts of water. Meanwhile, argon flow (Ar,

BOC, Pure Shield Argon) and magnetic stirring were applied and maintained throughout the

synthesis. Silver nitrate (AgNO3, 25 mM, Sigma-Aldrich, >99%) and poly(vinyl pyrrolidone)

(PVP, Sigma-Aldrich), with an average molecular weight Mw≈360k, were dissolved in EG

(3.5 mL) by magnetic stirring in the dark. The molar ratio of PVP to AgNO3 in the final

S2

reaction volume was 1.5 and the concentrations of PVP were calculated in terms of the

repeating unit. To remove oxygen, the AgNO3/PVP/EG solution was purged with Ar for 30

minutes. The AgNO3/PVP/EG solution (3.5 mL) was added to the reaction flask drop-wise.

After injection, the reaction mixture was refluxed at 160 oC and went through a number of

color changes until the mixture became stable at approximately 90 min. The reaction was

quenched by cooling the flask in a room-temperature water bath. The reaction mixture was

transferred to a centrifuge tube and diluted with acetone 5 times by volume. The AgNWs

were collected by centrifugation at 4500 rpm for 10 min. The washing process was repeated

by repeated cycles of centrifugation with ethanol and three times with deionized water (DI-

H2O), to ensure that residual EG, PVP and unreacted Ag+ ions were removed. To confirm that

most Ag+ ions had been removed, their concentration in the synthesis product was measured

by Inductively Coupled Plasma–Optical Emission Spectroscopy after ultrafiltration and was

found to be under the ICP-OES detection limit (i.e. <0.6 ppb). Finally, the sample was

dispersed in deionized water (5 mL) and stored in a sealed glass container at 4 oC in the dark,

to avoid exposure to contaminations and reactions induced by the ambient atmosphere (e.g.

sulfidation due to gaseous hydrogen sulfide (H2S), carbonyl sulfide (OCS) and carbon

disulfide (CS2) in the atmosphere). All the following experiments were performed using a

single batch of AgNWs.

Commercial AgNWs are available from at least 9 companies, with dimensions that

range between 20-200 nm in diameter and 2-200 μm in length.5 Therefore, the diameter of the

AgNWs synthesized for this work is at the average of this range while their length is at the

lower-end of the range. Most of previous in vitro and in vivo studies on AgNWs have been

performed on commercially available AgNWs.6-8 For example, the recent work by Silva et al.

compared the in vivo pulmonary effects post instillation of commercial AgNWs of two

different dimensions (“Short” AgNWs, length 2.0 μm, diameter 33.1 nm and “Long”

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AgNWs, length 20.8 μm, diameter 64.7 nm).6 Both AgNWs produced dose-dependent

inflammation indicative of foreign body responses in the lung, but different inflammatory

responses depending on AgNW length or higher dissolution rates by the smaller AgNWs.

Details about AgNW fabrication methods are rarely disclosed by manufacturers and few

characterization data of the products are provided. However, differences in the synthesis

procedures, AgNW dimensions or capping agents may lead to differences in dissolution rates,

agglomeration kinetics and ultimately in toxicological responses.9 In this work, we chose to

produce our AgNWs in house in order to have a full control over their physicochemical

properties, which were thoroughly characterized.

Animal LLF extraction:

Male rats were anesthetized by injection of a lethal dose of ketamine/xylazine, and

then sacrificed by exsanguination. Bronchoalveolar lavage (BAL) was collected using

buffered saline (1x-10mL wash). Cells were removed from the BAL by centrifugation

(300xg, 10 minutes at 4°C). The supernatant (2mL) was utilized to fractionate small

aggregate (SA, supernatant) and large aggregate (LA, pellet) portions of BAL. The LA was

re-suspended in 0.9% saline. Protein content for both the small (31.5µg/mL) and large

(166.1µg/mL) aggregate fractions was determined by the bicinchoninic acid assay (BCA),

Thermo Scientific (Rockford, IL, USA). Phospholipid content of the large aggregate fraction

was assessed by determining the concentration of organic phosphate (1.57µg/µL).10

This protocol was approved by the Rutgers University Institutional Animal Care and Use

Committee (IACUC) (Protocol Number: 06-028). The study was conducted in accordance

with the recommendations in the Guide for the Care and Use of Laboratory Animals of the

National Institutes of Health.

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AgNW incubations:

AgNWs were incubated at a concentration of 25 μg/mL from the original stock

solution (on a Ag atom basis as determined by ICP-OES) in a temperature-controlled dri-

block incubator at physiological temperature (37 oC) for 1 hour up to 336 hours (2 weeks) in

the dark. This dose was selected in order to provide direct comparisons with our previous

work on spherical AgNPs11 and on the in vitro effects of AgNWs on human alveolar

epithelial cells.12 Accurate dosimetry in laboratory evaluations of the effects of inhaled

particles has been a subject of concern, but very few data on AgNMs are currently available

to evaluate realistic occupational and consumer exposures.13 In one study, published

nanomaterial concentrations, including AgNPs, measured in air in manufacturing and

research and development laboratories were reviewed, to identify input levels for estimating

the nanomaterial mass retained in the human lung using the multiple-path particle dosimetry

(MPPD) model. Model results were then converted (using the surface area and volume

delivered in different types of cell culture well plates) to solution mass concentrations for in

vitro testing. For AgNPs, alveolar retention for a working-lifetime (45 years) exposure

duration was similar to higher concentrations (~ 50-200 µg/mL), tested in in vitro studies in

the literature. The alveolar retention for a 24 hour exposure duration was equivalent to lower

doses (~ 0.1-1 µg/mL) previously tested. Therefore, a dose representative of the lower-end of

those previously tested in in vitro studies of AgNMs was selected for this study,14 whose

primary aim is to deconvolute the effects of individual components of the LLF on the

physicochemistry of AgNWs.

The presence of complexing anions such as Cl- or S- is expected to lead to the

precipitation of insoluble silver species15, which would confound the measurement of free

Ag+ ions, therefore non-interacting perchlorate buffers were selected as the dispersion

medium. AgNWs were incubated in Sodium Perchlorate (NaClO4•H20, Sigma-Aldrich,

S5

>99%) (0.1 M) and the pH of the buffers was adjusted to 7 or 5, using either Perchloric Acid

(HClO4, Sigma-Aldrich, 70%, 99.999% trace metals basis) or Sodium Hydroxide (NaOH,

Sigma-Aldrich, anhydrous, 99.99%). These pH values were selected to simulate characteristic

environments found in the lung. The interstitial and alveolar extracellular fluids have a pH of

∼7.416 while the cell cytoplasmic pH is ∼7.2. In the endocytic pathway of the cells, the pH

decreases progressively from the early endosomes (pH∼6.5) to late endosomes (pH<6.0) and

ultimately lysosomes (pH<5.5).17

To study the effect of each component of the lung surfactant on the stability of

AgNWs, various combinations of these components were incubated together. Human

surfactant consists mostly of phospholipids, with the most abundant being DPPC, therefore

DPPC was used to study the effect of phospholipids. The effect of the two hydrophobic

surfactant-associated proteins, SP-B and SP-C, was investigated using Curosurf®. Curosurf®

is a natural surfactant, prepared from porcine lungs and used for the treatment of endogenous

pulmonary surfactant deficiencies by intratracheal administration. It contains almost

exclusively phospholipids but also about 1% of SP-B and SP-C. Finally, the role of SP-A and

SP-D on the stability of AgNWs was studied by adding the small aggregate (SA) fraction of

lung surfactant extracted from rat lungs. SA contains the more soluble components of the

lung lining fluid, such as the non-specific lung proteins IgG and albumin, but also the

collectins SP-A and SP-D. Atochina et al. showed that, in the bronchoalveolar lavage of

control healthy mice, over 90% of phospholipids, 100% of both SP-B and SP-C and about

60% of SP-A, could be found in the LA fraction. In contrast, the majority of SP-D and the

remaining SP-A were found in the SA fraction.18

A stock solution of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Sigma-

Aldrich, semisynthetic, ≥99%) (10 mg/mL) in DI-H2O was prepared by magnetic stirring

overnight. DPPC, Curosurf® (poractant alfa, Chiesi Farmaceutici, S.p.A.) and LA were used

S6

at amounts that correspond to a total lipid concentration of 100 μg/mL. SA was used at

amounts that correspond to a total protein concentration of 100 μg/mL. These concentrations

were chosen to correspond to the average values observed in the surfactant obtained from the

bronchoalveolar lavage fluid of healthy persons.19 For the phospholipids that constitute most

of pulmonary surfactant, the critical micelle concentrations (CMC) fall within the range of

10−10 to 10−9 M.20 For DPPC, for instance, a CMC of 10−10 M is equivalent to 7.34×10-5

μg/mL, therefore the formation of micelles is expected in our experiments. However, in

essentially all of the in vitro studies, and at estimated physiological concentrations,

pulmonary surfactant is well above its CMC.20

Scanning Electron Microscopy (SEM):

The morphology and size distribution of the AgNWs were characterized using a LEO

1525 Field Emission Gun Scanning Electron Microscope (FEG-SEM, Carl Zeiss Microscopy

GmbH, UK). The SEM was operated in secondary electron mode at an accelerating voltage

of 5 kV, using the InLens detector. Samples were prepared by drop-casting aliquots of the

AgNW suspensions on a piece of silicon wafer and dried under ambient conditions in a fume

cupboard and in the dark. Samples were stored under vacuum and in the dark. The size

distribution of the AgNWs was characterized using SEM images and ImageJ software

(http://rsb.info.nih.gov/ij/).

Transmission Electron Microscopy (TEM) Sample Preparation:

To prepare TEM samples from the incubated AgNWs, aliquots were removed at each

time point and, after washing three times with DI-H2O to remove excess salts or organic

surfactant, they were drop cast on 300 mesh holey carbon film TEM grids (TAAB) in the

dark. The grids were blot-dried with filter paper and were immediately placed under vacuum

S7

and in the dark to avoid reactions induced by ambient atmosphere. The grids were imaged

within a period of 2 weeks. For TEM samples prepared from as-synthesized AgNWs and

imaged after a storage period of up to 3 months under vacuum, no changes in their

physicochemical properties were detected (more than 100 wires analyzed, Figure S1). To

enhance phospholipid contrast, samples were positively stained with 2% uranyl acetate in

water.

Figure S1. TEM images of a sample prepared from as-synthesized AgNWs, imaged after a 3-

month storage period under vacuum and in the dark.

TEM:

Bright field transmission electron microscopy (BFTEM), high resolution transmission

electron microscopy (HRTEM) and high angle annular dark field scanning transmission

S8

electron microscopy (HAADF-STEM), combined with selected area electron diffraction

(SAED) and energy-dispersive X-ray spectroscopy (EDX) were carried out using a JEOL

JEM-2100F fitted with an EDX detector (Oxford Instruments). The scattering intensity in

HAADF-STEM is proportional to Zn (n ~ 2), therefore this technique is highly sensitive to

atomic number (Z) variations within the sample. An accelerating voltage of 200 kV was used

for both TEM and STEM experiments. For STEM experiments, the inner and outer HAADF

collection angles were 150 and 400 mrad, respectively, and the probe diameter was <1 nm.

Light Microscopy (LM):

Aliquots were removed from the incubated AgNWs, placed on a clean glass slide,

coverslipped and immediately observed in reflectance mode, using a Leica DM2500 with

reflected axis LED light source and a Leica DFC295 camera. The objective lenses used were

an Olympus 50x/aperture 0.75 and an Olympus 100x/aperture 1.25.

Statistics:

The LM/SEM/TEM images presented and the statistics on particles were obtained by

viewing several AgNWs (n≥100 for each sample), from multiple areas of three samples

prepared under identical conditions.

Inductively Coupled Plasma–Optical Emission Spectroscopy (ICP-OES):

To determine the amount of free Ag+ ions released from the incubated AgNWs at 1

hour up to 336 hours, aliquots were collected at each time point and centrifuged at 13000×g

for 10 minutes through 2 kDa centrifuge membranes (Sartorius Stedim VIVACON 500) to

remove the AgNWs. The amount of Ag+ in the filtrates was measured by Inductively Coupled

Plasma–Optical Emission Spectroscopy (ICP-OES, Thermo Scientific, UK) with a silver

S9

detection limit of 0.6 μg/L. Each experiment was repeated three times and the results are

given as the mean and standard deviation of the three repeats. The method of centrifugal

filtration used leads to 100% of Ag+ ion recovery, as we have previously demonstrated in a

control experiment where a solution of AgNO3 in DI-H2O was subjected to filtration and the

total amount of Ag+ added was measured by ICP-OES in the filtrate.21 To confirm that

secondary AgNPs are also filtered by the centrifuge membranes, AgNWs were incubated at

pH7 for 24 hours. The UV-Vis spectrum of the sample was collected before and after

filtration, with no peaks observed in their UV-Vis after filtration (Figure S2).

300 350 400 450 500 550 600 650 7000.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Abso

rban

ce (a

.u.)

Wavelength (nm)

Before Filtration After Filtration

Figure S2. UV-Vis spectra of AgNWs incubated at pH7 for 24 hours, before and after

centrifugation at 13000×g for 10 minutes through 2 kDa centrifuge membranes.

S10

Figure S3. (a-b) STEM-EDX spectrum collected from the circled area imaged in Figure 4 h.

(c) HRTEM image and (d) SAED pattern collected from AgNWs incubated with DPPC at

pH7 (same sample as Figure 4 h-l). The interplanar spacings in the SAED pattern correspond

to the (111), (200), (220) and (311) planes of the bcc form of bulk Ag (ref. # 01-087-0597),

confirming that the crystallography of AgNWs incubated with DPPC has not changed.

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