Nuclear Localisation Signal (NLS) is a peptide sequence that tags certain proteins for nuclear transport. By conjugating NLS to gold nanoparticles (GNPs), this should (in theory) result in the GNPs being transported to the nucleus. This can then be detected using photothermal microscopy. However, actually putting this into practice is slightly harder. Due to the abundance of positively charged residues in the NLS sequence they can cause the nanoparticles to aggregate.
Before producing a large batch of GNPs conjugated to NLS, I first needed to determine how much NLS could be added without causing aggregation. To find this I made a range of ligand solutions of NLS and CALNN, ranging from 100% NLS right down to 0.5% NLS. Whether or not the GNPs aggregate can be determined both visually (by observing a colour change from red to blue) and by using a UV-vis spectra. Solutions of 1% and 0.5% NLS appeared to be stable, however these then aggregated in larger preparations. Initially GNPs with 0.01% NLS were used for microinjection, however after revisiting the NLS experiment I found that 0.25% NLS was also stable.
Three sets of microinjection were done using different preparations of GNPs conjugated to NLS. The first was a 57nM solution of GNPs, with 0.01% NLS. The first cell imaged appeared promisingly full of particles, however less promisingly the following cells had little or no evidence of being successfully injected (see images below).
After this, I revisited the GNP conjugation to NLS and prepared a more concentrated sample (130nM) with a higher percentage of NLS (0.25%). These were then injected and photothermally imaged. Although the particles seemed to inject well and did not appear to have aggregated whilst being prepared, when the cells were imaged large aggregates were seen around or on the cells. This may have been caused by the presence of NLS on the nanoparticles surface, causing them to aggregate as they became more concentrated.
The images obtained after using the more concentrated GNPs showed that the particles could potentially aggregate if concentrated too much. I then once again prepared another batch of GNPs conjugated to 0.25% NLS without concentrating them very much. The images taken after these were injected showed large numbers of particles successfully injected into multiple cells.
Although by the third set of particles the images obtained showed large numbers of GNPs present in the cell, there was no real evidence that tagging the nanoparticles with NLS resulted in nuclear transport. There are many factors that may have caused this, from not incubating the cells for long enough before fixing them, to the size of the nanoparticles restricting them. The latter can be addressed by using smaller nanoparticles, which is hopefully what I will try to do next.