The role of surfactant proteins A and D (SP-A and SP-D) in lung clearance and translocation to secondary organs of inhaled nanoparticles was investigated by exposing SP-A and SP-D knockout (AKO and DKO) and wild type (WT) mice nose-only for 3 hours to an aerosol of 20 nm gold nanoparticles (AuNPs). Animals were euthanised at 0-, 1-, 7- and 28-days post-exposure. Analysis by inductively coupled plasma mass spectrometry (ICP-MS) of the liver and kidneys showed that extrapulmonary translocation was below the limits of detection. Imaging of the lungs by laser ablation ICP-MS confirmed the homogenous distribution of AuNPs. Coherent anti-Stokes Raman Scattering, Second Harmonic Generation and Two-Photon Fluorescence imaging were applied for semi-quantitative analysis of the uptake of AuNPs by alveolar macrophages and found uptake increased with time post-exposure, peaking after 7 days, and with the largest increase in uptake being in WT mice. Single particle ICP-MS allowed particle counting and sizing of AuNPs in the lungs showing that particle agglomeration following deposition within the lung was greater for the wildtype than the knockout models, indicating a role for SP-A and SP-D in agglomeration, however, any effect of this on overall lung clearance was minimal. For all groups, the Au (mass) lung burden initial clearance half-time was approximately 20-25 d, however, the AuNP (particle number) lung burden clearance half-time was shorter at approximately 10 days. In general terms, differences between the results for the three models were limited, indicating the preferential clearance of smaller particles from the lung.
Keywords: Raman; SP-A; SP-D; Single particle ICP-MS; lung clearance; lung surfactant; nanoparticles.