Influence of lung secretions on the uptake of latex nanoparticles by human alveolar type I epithelial cells

Thomas Potter, Andrew Thorley, Teresa Tetley. National Heart and Lung Institute, Imperial College London, Lung Cell Biology, SW3 6LY London, United Kingdom.

The use of engineered nanoparticles as carriers for inhaled drug delivery is increasing rapidly due to their desirable properties, namely large surface area to volume ratio, ability to adsorb compounds and most importantly, their nano-size. Approximately 50% of inhaled nanoparticles preferentially deposit in the alveolar region which has a surface area of over 150m2 and a rich blood supply making it an excellent site for absorption. However, the physicochemical properties that make them ideal for drug delivery may also cause toxicity.

We hypothesised that the size and surface modification of latex nanoparticles would affect how they interact with proteins in human lung secretions and thus their uptake by the alveolar epithelium.

To investigate whether proteins present in lung secretions are adsorbed on to the surface of nanoparticles, 50nm and 100nm fluorescently-labelled latex nanoparticles that were either unmodified, carboxyl-modified or amine-modified were incubated with human lung lining liquid (HLLL) for 3 hours. Protein adsorption was analysed using 1D gel electrophoresis, and immunoblotting for surfactant proteins A and D (SP-A and SP-D). Nanoparticle agglomeration was visualised using Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS). Uptake of NPs by immortal human alveolar type I epithelial (TT1) cells was measured using fluorescence microscopy.

All types of nanoparticle adsorbed proteins from the HLLL. Western blotting demonstrated that surfactant proteins A and D bound to all nanoparticles but more so to carboxyl-modified nanoparticles compared to unmodified and amino-modified. Incubation with lung secretions also increased the agglomeration of 50nm and 100nm amino-modified nanoparticles compared to the respective unmodified and carboxyl-modified nanoparticles as demonstrated by DLS and TEM.

To investigate how adsorption of proteins affects uptake of nanoparticles by TT1 cells, confluent monolayers were exposed to 40µg/ml nanoparticles in the presence and absence of HLLL fluid for 4 hours. The degree of uptake of nanoparticles was size-dependent and that incubation with BAL fluid significantly increased uptake of all types of nanoparticle (P<0.01). Significantly more 50nm nanoparticles were internalised than 100nm nanoparticles (approx. 1.3 fold; P<0.05) for all surface modifications (MFI values: carboxyl 91vs 65; amino 107 vs 89; unmodified 49 vs 45 respectively). Furthermore, incubation with HLLL fluid potentiated the uptake of carboxyl-modified nanoparticles significantly more than amino-modified and unmodified nanoparticles (P<0.05).

These results demonstrate that both size and surface modification of nanoparticles critically affect how they interact with protein secretions at the lung surface and their subsequent internalisation by the alveolar epithelium. These are important considerations in design of NPs for pulmonary drug delivery.