Characterising nanoparticles in complex biological media

Progress in the application of nanotechnology within medicine has been limited in part due to the difficulties in understanding and predicting the behaviour of nanoparticles in complex biological media. How nanoparticles disperse in biological media and the interactions that occur at the bio-nano interface dramatically influence their subsequent biological function. Techniques to measure and monitor these behaviours are usually bulk techniques, however these can be limited by the more complex nature of biological environments which routinely contain a number of bio-macromolecules e.g. vitamins, proteins and salts. As such, high spatial resolution analysis through electron microscopy has been investigated as an alternative to more accurately characterise nanoparticles in biological fluids. However, electron microscopy itself can be limited by the ultra-high vacuum requirements which mean samples must be dried before analysis. This necessitates the development of in situ techniques in order to characterise nanoparticle suspensions in the representative, native hydrated state. Using cryogenic transmission electron microscopy (cryo-TEM) a sample can be analysed in the frozen, hydrated state. However, typically this is limited to imaging alone. In this thesis, the use of analytical scanning TEM (STEM) to characterise nanoparticle dispersions captured in a layer of vitreous ice is demonstrated using both STEM energy dispersive X-ray spectroscopy and electron energy loss spectroscopy under cryogenic conditions. A noticeable difference in damage to the surrounding vitreous ice was observed between conventional TEM (CTEM) and STEM where damage occurred at much higher electron fluences in STEM (<2000 e-/Å2) compared to CTEM (<100 e-/Å2). Applying these techniques to characterise nanoparticles dispersed in cell culture media showed that incorrect specimen preparation or analysis where a significant raise in pH level is caused can induce an artefactual, nanoscale, calcium phosphate-rich, amorphous coating on nanoparticles dispersed in cell culture media. Recommendations to prevent this are given which will prevent any specimen preparation artefacts that could drive alterations in the in vivo or in vitro function of nanoparticles. For nanoparticle dispersion analysis automated electron microscopy imaging and analysis of plunge frozen vacuum dried nanoparticle suspensions was shown to be a viable alternative to dynamic light scattering for quantification of nanoparticle agglomerates in biological dispersants. Using two simple freeware codes, CellProfiler and Ilastik automated image analysis was achieved and validated for both monodisperse and agglomerated nanoparticle systems. Finally, cellular uptake studies assessed the biological effect of two gold nanoparticles coating with PEG and either terminated with a positive NH2 or neutral OMe group, on blood cells. No significant differences in cell uptake, geno- and cyto- toxicity or immune response was observed for the two nanoparticle types. Protein corona analysis by sodium dodecylsulfate - polyacrylamide gel electrophoresis indicated a comparable hard corona composition for both particles. Preliminary work identified annular bright field-STEM as a potential pathway to image the protein corona. This characterisation indicated possible inter-particle variation in the presence of a corona around particles within an individual suspension. Overall, the results reported show in situ cryo-analytical S/TEM is a powerful tool to characterise nanoparticle dispersions in complex biological media in order to further the understanding of complex bio-nano interactions and nanoparticle dispersion behaviour that ultimately control biological function.