Review of 'The Spherical Nucleic Acids mRNA Detection Paradox'

This article by Lévy et al aims to debunk the claims that SmartFlares can detect mRNA in the cytoplasm of cells. The authors briefly summarise the current state of the literature accurately: there is no cell biological reason for thinking that these nanoparticles should actually work. Good evidence for endosomal escape has not been documented so far. They then set out to characterise SmartFlare activity and conclude that these particles do not work as advertised. This is an important, but thankless, task. Important because it will prevent others from wasting time and resources on something that doesn't work. Thankless because it can often take a great deal of effort to overturn (attractive) ideas.

As a cell biologist, one area for improvement in this paper that I see would be to quantify more cells that have been doubly labelled to better characterise the compartment that the SmartFlares end up in. Figure 4 shows these experiments, but we only see one or two cells, with a single Mander's coefficient. I would prefer to see more cells quantified. Maybe the location of the vesicles containing SmartFlares is variable, but this could be better documented than it is now. Related to this:

"We labelled the endocytic pathway using a fluorescently-labelled 10 kDa dextran, both to show that constitutive uptake was occurring in our system, but also to label all of the compartments of the pathway. As expected, the dextran labelled every cell in the field with homogeneous intensity puncta (Figure 4B). There were approximately the same number of vesicles per cell when corrected for cell size. Interestingly, the dextran and SmartFlares rarely labelled the same compartments (Figure 4C) even at a 2 hour time point (Supplementary Figure 1).

As the dextran may be excluded from receptor-mediated endosomes (by unknown mechanisms) we also used immunofluorescence to label the recycling (transferrin-receptor positive) and terminal
(lysosomal) compartments of the endocytic pathway (Figure 4D-F and G-I respectively). Once again, the SmartFlares showed little overlap with either of these compartments (Figure 4C,F,I, Manders’ coefficients inset), suggesting a parallel but largely non-overlapping compartment."

I don't agree with this interpretation. From the examples shown it looks like there is significant colocalisation with fluorescent dextran (Fig 4C and Supp Fig 1). This condition also has the highest Manders coefficient, compared to Tf and LAMP1 staining. Where there is also some overlap, albeit limited.

It seems clear from the work presented in earlier figures that the particles are retained in vesicles. There's partial overlap with these compartments at the resolution of light microscopy. Some more quantification would be good to try to nail down where the SmartFlares end up. 

Minor
I thought to improve presentation that the figures could do with more labelling and less lettering. For example on Fig 4, the columns could be labelled SmartFlares, Stain and Merge (l-r). The rows could be labelled with 10K dextran, TfR and LAMP-1 (top to bottom). This means that the figure can be understood by just looking at it. 

There is no label on the y-axis in Fig 3E.

Very minor
1. I'm not a big fan of using number of Web of Science search results as an argument (Introduction). The number of papers on Gold Nanoparticles may be increasing since 2007, but then so are the number of papers on anything. It needs to be normalised to be meaningful. It's also a shame that only 5 papers have cited Harford et al., but it's an old paper, maybe people are citing reviews that cover this paper instead?
2. Technai is spelled Tecnai
3. Extra I J K below the panels in Fig 4 need to be removed.