Microscopic Analysis of the Interaction of Gold Nanoparticles with Cells of the Innate

Microscopic Analysis of the Interaction of Gold Nanoparticles with Cells of the Innate Immune System.

César Pascual García1+*, Vadim Sumbayev2+, Douglas Gilliland1, Inna M. Yasinska2, Bernhard F. Gibbs2, Dora Mehn1, Luigi Calzolai1 and François Rossi1

1Nanobiosciences Unit, Institute of Health and Consumer Protection, Joint Research Centre Via Fermi TP203, 21027 Ispra (Italy)

2Medway School of Pharmacy, University of Kent, Anson Building, Central Avenue, Chatham Maritime, Kent ME4 4TB, United Kingdom

SUPLEMENTARY INFORMATION
Nanoparticle synthesis.

2.1.1. 5 nm AuNP

Au NPs were produced by NaBH4 (Sigma) reduction of a starting solution of sodium citrate (Sigma) (2.5 mM) and HAuCl4 (Sigma) (0.5mM) in water. Typically 100 mL of the starting solution was cooled in an ice bath to 4°C before the reduction was initiated by the addition of 1 mL of freshly prepared ice-cold NaBH4 solution (0.1 M) under rapid stirring. Au NPs formation was observed to occur immediately as shown by the rapid color change from pale yellow to wine red.

2.1.2 15 nm AuNP

HAuCl4 (0.5 mM) in water was rapidly heated to 97ºC in a microwave heating system (Discover S by CEM) and allowed to equilibrate for 5 minutes. The reaction was initiated by injection of sufficient sodium citrate (0.1 mM) to produce a final citrate concentration of 2.5 mM. The reaction mixture was then kept at 97 ºC for 20 minutes after which the reaction vessel was rapidly cooled to room temperature.

2.1.2 35 nm AuNP

100ml of HAuCl4 (0.5 mM) was heated to 97 ºC in a microwave heating system and then allowed to stabilize for 5 minutes. The pH of this solution was then increased to >10 by the addition of 1.1 ml of NaOH (0.2 M). At this point the reduction reaction was initiated by injecting sodium citrate (0.01mM) solution to produce a final citrate concentration of 0.5 mM. The higher pH of this mixture ensured that the reduction of gold by citrate proceeds slowly with the formation a smaller number of larger particles. The solution was maintained at 97 ºC for 20 minutes at which point the solution was seen to be a slightly purple/pink color indicating the partial conversion the gold salt into gold NPs. The solution was then cooled to 90 ºC under stirring at which point 1 ml of NaOH (0.2 M) then 2.5 ml of NH2OH∙HCl (0.1 M) were injected into the solution. The addition NH2OH∙HCl promotes the reduction of the remaining gold ions in solution selectively on the surface of existing gold nanoparticles so increasing the size of the existing particles rather than nucleating new particles. The second addition of NaOH serves to adjust the pH of the final solution to value of around 6.5-6.8 which is comparable with the other two synthesis methods.

2.3. Cell milling

In our experiments we used a double beam scanning electron microscope FEI-Nova nanolab 600I equipped with FIB and Gas injectors with precursors of platinum, gold, silicon dioxide and carbon milling. Our system is equipped with a traditional secondary and in-lens detectors for traditional and immersion imaging of the scattered electrons, as well as X-ray analyser used for EDAX with element spectral resolution and sensitivity down to carbon. We also used an STEM detector for transmitted electrons with 8 channels.

As explained in the body of the article after detecting a correct shaped cell (Figure sup info 1 A), we firstly scanned it using the transmission mode to detect Au NPs using 30 KeV HV. After identifying the position of the NPs, we explored the surface of the cell using lower HVs that ranged from 5 to 25 KeV. If a NP detected in transmission was absent from the surface, we proceeded to search for it by performing a cut, using FIB milling, which permitted us to assess the potential cellular uptake of NPs. Before the cut, and in order to protect the region of the cell containing the NPs, we deposited a protective metallic layer on top using Pt-GIS activated with the electron gun to avoid curtain effect (Figure sup info B). To avoid the charging effects, the Pt-GIS was activated with the same energy we used to visualize the surface. The cut was made using the smallest aperture of our FIB at 1.5 pA current to avoid damage caused by the FIB to the cells. The angle between our SEM and FIB guns is 52º (Figure sup info C). For the cut, the sample was tilted perpendicular to the FIB. In this configuration it is possible to measure the height of the section of the cell corresponding to the position of the NPs detected in transmission. We used this parameter to limit the cut. This configuration allowed us to perform the cut and image the sample as seen in Figure sup info B.

Sup information figure A) Scanning electron picture of a cell incubated with 35 nm NPs at 00. B) Scanning electron picture of the same cell after the FIB cut at 520. The AuNPs found are marked by the yellow arrow. C) Schematic representation of the microscope chamber.