On 2015 Jul 15, Raphael Levy commented:

Thanks Harald for commenting on my blog post about SmartFlares / Nanoflares with reference to this paper.

I reproduce the conversation below. I hope it continues and others join in.

Raphael

"Hi everybody, as the correspending author of a Stem Cell paper in which we have used the SmartFlares on different pluripotent cells of human, murine and porcine origin I want to reply to two of the above mentioned questions.

Why do we see a signal at all in the scramble control? I think one cannot expect a negative control which does not produce a fluorescent signal at all. The fluorophore may not be quenched by 100% and may be subject to degradation, especially when applied for a longer time (two days or more). Nevertheless, within 16 to 24 hours after the application of the nanoparticles we see a clear-cut difference of fluorescence intensity when comparing scramble control and gene-specifc Smart Flares. http://www.ncbi.nlm.nih.gov/pubmed/25335772

We believe that this difference is reliable and specific. We have selected freshly reprogrammed murine iPS cells based on their Nanog-specific fluorescence intensity in situ. In downstream experiments we could confirm that only colonies with a high fluorescence intensity expressed higher amounts of endogenous pluripotency factors and showed a superior capacity to differentiate. Therefore, we belive that these functional data strongly support the idea that the fluorescence intensity was indeed correlated to a specific interaction with the Nanog mRNA in these clones.

Why do different cells take up varying amounts of SmartFlares? I think this difference is not surprising as the nanoparticles are engulfed by endocytosis. This process is influenced by the cell type, the differentiation status and the cellular ability to perform phago- and macropinocytosis. Therefore, we think that a uniform uptake rate cannot be expected."

I replied "Hi Harald

Thanks again for commenting here and sorry for the delay in replying. It is interesting that you see some differences but the big question that remains is how could the technology possibly work?

It can only work if the particles escape endosomes, but: 1) there is no reason why they should, 2) this problem is not discussed in the original publication introducing the technology, 3) there is no direct evidence in the literature that it happens, and, 4) all the data we are accumulating indicates that the particles are indeed in vesicular compartments (more on this soon on the open notebook as we have just had our cell electron microscopy results this week).

The images shown in your articles are low mag overviews of many cells and therefore the resolution does not allow to discuss any cellular localization. Do you have any higher resolution images that you could share? Do you have any (direct) evidence and/or proposed mechanism for endosomal escape?

The unequal distribution of uptake (cell to cell variability) is also a big concern. I don鴠believe that it relates to differences between rate of uptake of different cells. Such differences would average over an 18 hour period and they should also be seen in the dextran uptake. A possible interpretation would be some degree of nanoparticle association/aggregation before interaction with the cells (this is to be tested experimentally).

Raphael"

On 2015 Jul 15, Raphael Levy commented:

Thanks Harald for commenting on my blog post about SmartFlares / Nanoflares with reference to this paper.

I reproduce the conversation below. I hope it continues and others join in.

Raphael

"Hi everybody, as the correspending author of a Stem Cell paper in which we have used the SmartFlares on different pluripotent cells of human, murine and porcine origin I want to reply to two of the above mentioned questions.

Why do we see a signal at all in the scramble control? I think one cannot expect a negative control which does not produce a fluorescent signal at all. The fluorophore may not be quenched by 100% and may be subject to degradation, especially when applied for a longer time (two days or more). Nevertheless, within 16 to 24 hours after the application of the nanoparticles we see a clear-cut difference of fluorescence intensity when comparing scramble control and gene-specifc Smart Flares. http://www.ncbi.nlm.nih.gov/pubmed/25335772

We believe that this difference is reliable and specific. We have selected freshly reprogrammed murine iPS cells based on their Nanog-specific fluorescence intensity in situ. In downstream experiments we could confirm that only colonies with a high fluorescence intensity expressed higher amounts of endogenous pluripotency factors and showed a superior capacity to differentiate. Therefore, we belive that these functional data strongly support the idea that the fluorescence intensity was indeed correlated to a specific interaction with the Nanog mRNA in these clones.

Why do different cells take up varying amounts of SmartFlares? I think this difference is not surprising as the nanoparticles are engulfed by endocytosis. This process is influenced by the cell type, the differentiation status and the cellular ability to perform phago- and macropinocytosis. Therefore, we think that a uniform uptake rate cannot be expected."

I replied "Hi Harald

Thanks again for commenting here and sorry for the delay in replying. It is interesting that you see some differences but the big question that remains is how could the technology possibly work?

It can only work if the particles escape endosomes, but: 1) there is no reason why they should, 2) this problem is not discussed in the original publication introducing the technology, 3) there is no direct evidence in the literature that it happens, and, 4) all the data we are accumulating indicates that the particles are indeed in vesicular compartments (more on this soon on the open notebook as we have just had our cell electron microscopy results this week).

The images shown in your articles are low mag overviews of many cells and therefore the resolution does not allow to discuss any cellular localization. Do you have any higher resolution images that you could share? Do you have any (direct) evidence and/or proposed mechanism for endosomal escape?

The unequal distribution of uptake (cell to cell variability) is also a big concern. I don鴠believe that it relates to differences between rate of uptake of different cells. Such differences would average over an 18 hour period and they should also be seen in the dextran uptake. A possible interpretation would be some degree of nanoparticle association/aggregation before interaction with the cells (this is to be tested experimentally).

Raphael"