Lahm et al have published a “peer reviewed scientific video” at the video journal JoVE. Below is a copy of a comment I left at PubPeer. Comments welcome here or at PubPeer.
The video is fairly similar in tone and message to the EMD Millipore advertising video (https://www.youtube.com/watch?v=CiSC2B8hl1E). It does not address the essential paradox that underpins this technology: how do the particles escape endosomes? and, if they don’t, how do they detect cytosolic mRNA?
In our experience, the particles remain trapped in endosomes and the fluorescence increase is most likely due to degradation of the oligonucleotides by nucleases inside endosomes rather than mRNA detection. We will publish this work in the next few days and I will add a link here.
You can already find our data and analysis on our open science notebook, data repository and blog.
There are also three relevant PubPeer threads:
https://pubpeer.com/publications/46B30327D98DFCA359C7EC7BD624AA
https://pubpeer.com/publications/25CC01C366B9593D1686A78B52461F
https://pubpeer.com/publications/904628FEFA9EED0738718BFF169180
Incidentally, a similar question regarding the limit between advertising and scientific publication was asked by George McNamara when he noted that a conflict of interest statement should have been included in the Sticky-flares PNAS paper.
Rapha-z-lab 
The film is very convincing that you may be right. It is actually quite creepy. “It’s smart.” On the other hand, Zeljka working with TAT modified particles (the paper that you did not want to be on), has exactly observed this sequence of events including the reformation of particle-filled vesicles, probably by autophagy. Antonis, by the way, told me that he uses this product and “it works”. If you are right, it would not, at least if if you do a negative control experiment, which I assume he must have done. M
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I have invited Antonis to join the conversation.
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Hi All,
Raphael thanks for sending me your web link. We work on the nanoflares the last 3 years and we prepare now our first papers for submission. We do not buy nanoflares. We make them ourselves. We have done several controls positive and negative and we concluded that the nanoflare technology works very well (at least for the nanoflares that we make in the lab). Apparently many flares can escape from the endosomes and actually we noticed some localization in mitochondria for some specific sequences. We have not yet understood the mechanism of endosomal escape but we work on this too (1 phd student’s time is fully dedicated on this and a postdoc is helping). It is very weird the fact that they escape (since they are also negative charged) and we look forward to find the answer to this. Many controls is absolutely necessary when confocal is used otherwise you can get false results. Hope this helps.
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Hi Antonis, thanks for your comment. Very interesting. I am looking forward to see the data. How you done an extensive quantification by electron microscopy of the proportion of particles that escape endosomes? At which time point does this escape occurs? Our EM data show endosomal localisation:
http://cci02.liv.ac.uk/gallery/show_dataset/1945/
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Hi Raphael,
We are not able to do any quantification at this stage. TEM certainly is not a way forward for quantification though since you cut and observe only few very thin sections at a time.
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Quantification by TEM is possible though requires a very large effort; see a (very relevant) example and discussion here:
https://pubpeer.com/publications/BECD9A153DB5564060F49D684588B4
Even without going to such extreme, a comprehensive TEM study would give an idea of whether there is a significant proportion of particles outside endosomes.
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Hi Raphael,
Try to not rely on TEMs regarding your study. Use it only as a complementary technique. We do see particles in and out of endosomes. I think Amelie even saw few in mitochondria (if not mistaken). I asked her to reply to you.
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Thank you for advice Antonis (I assume this is still Antonis?). TEM is a very powerful technique to determine exactly the intracellular localisation with organelle specificity. In our study, it plays a role but is also complemented by extensive fluorescence microscopy as well as photothermal microscopy. You can find all of our data on the repository and all of our protocols on the open notebook. Looking forward to see your results and interpretation. Too bad that the publication process is once more a barrier to the free exchange of ideas and to scientific discussion…
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Hi Raphael,
just quickly to correct Antonios. We did indeed see some particles outside of endosomes (I haven’t done an extensive quantification as per the paper you mentioned yet though). However, the mitochondrial association we saw was based on fluorescence rather than TEM.
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@Antonis: also looking forward to reading the paper! Have you made any adjustments to the SNAs to decrease nuclease-mediated degradation in the endocytic pathway? We suspect, and the Mirkin Group have shown (doi:10.1021/ja503010a) this to be a big problem for SNAs delivered by endocytosis.
PS. Regarding mitochondrial localisation, would love to hear your thoughts on point 3a here: https://pubpeer.com/publications/25CC01C366B9593D1686A78B52461F
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Hi Dave,
In our systems (having the flare hybridised) we avoid enzymatic degradation. Please try not to make straight comparisons. Everything plays a role. I cannot give more technical details before we publish. However, my postdoc, Amelie, will check the blog and the link and reply to you. I am in rush going for teaching now.
All the best
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Hi Dave, without being able to go into too much detail: Yes, we have optimised the system such that we do not observe any noticeable intracellular degradation. Regarding the mitochondrial co-localization we have indeed also observed this (for several cell types) with Cy-dye labelled flare strands as discussed in the papers you mention in point 3a
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Hi Raphael,
Yes, as you mentioned, TEM is a good technique but only when you complement it with other techniques, as you discuss in your work. What I meant was not to rely only on TEMs for your studies.
Good luck with your experiments.
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