Discussion Points from SmartFlare Group Meeting

I recently discussed the SmartFlare project at a joint Sée and Lévy Group Meeting, partially extolling the virtues of Open Science but mostly focussing on the data acquired so far and some of the proposed experiments.

By Andreas E. Neuhold[CC BY 3.0], via Wikimedia Commons
Regarding the SmartFlare project, the three big questions that formed the basis of the talk were:

  • Why do only some cells take up SmartFlares?
  • Where are the SmartFlares?
  • Why are the VEGF and Scrambled SmartFlares fluorescent in puncta?

Below are some of the interesting points that were made during the meeting. Replies at the time (as best as I can remember them) in green:

  • In relation to the heterogeneity of SF uptake, it was asked whether the ‘dark’ cells may have taken up the SmartFlares but just lack detectable VEGF mRNA. This is a really interesting question as there’s not really any way to see the SmartFlares when they’re quenched using fluorescence microscopy. Multimode fluorescence and photothermal is a real option, however, even the Uptake Controls show heterogeneous uptake suggesting that this is unlikely to be a quenching/de-quenching effect.
  • Could possible quenching effects explain the same phenomenon. Would lower concentrations of SmartFlares actually show more signal? Possible, as this could affect controls as well as VEGF SmartFlares. I think this is unlikely, but again, photothermal would be a great way to ‘image’ the distribution of gold nanoparticles in cells. At this point, we’re following the manufacturer’s guidelines, so presumably it’s been extensively tested at this concentration.
  • Would longer time points show SmartFlares escaping from the vesicles? Again, a possibility and an easy experiment to do, but as stated above, we’ve been following the manufacturer’s guidelines which recommend 18-24h exposure.
  • May be worth trying a shorter treatment with chloroquine once particles have been taken up. Absolutely. The chloroquine literature is quite conflicted as regard whether the drug destabilises endosomes, prevents maturation or inhibits endocytosis all together. (UPDATE: See “Interpretation of the Chloroquine results” for more information)

Some other interesting points and random thoughts that came up during the meeting:

  • Microinjection of SmartFlares
    • This is, in my opinion, a really important experiment to do. None of the experiments so far hint that the SmartFlares work as advertised. It’s important to know if this is a problem with delivery or with the product. Microinjecting directly into the cytosol bypasses delivery, but will probably lead to massive variability in loading cell to cell.
  • Fluorescent spectrophotometer / FLiM measurements +/- DTT
    • Similar to the microinjection is some basic in vitro work to make sure that changes in fluorescence associated with de-quenching are even detectable. Putting the SmartFlares in a cuvette +/- DTT to strip the oligos should give us the dynamic range.
  • Quantitative texture analysis
  • Caveolin / Caveosomes
    • This one is going to get a post all to itself. A fascinating if somewhat confusing subject.
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2 thoughts on “Discussion Points from SmartFlare Group Meeting

  1. Hi Dave

    Thanks for the lab meeting and for keeping this open science experiment very much alive (I hope we will be soon having more projects coming through this open notebook). In the spirit of openness, I thought I would comment here on how we should try to wrap up this investigation in the next 1-2 months. Largely follows what you write above, but trying to focus a bit.

    An exhaustive investigation of how the smartflares enter cells [which would probably also be cell-type dependent] is beyond the scope of our study. We might not be able to answer in detail all of the questions raised by your data.

    I would focus the discussion as follows.

    I would start by the elephant-in-the-room of the smartflare litterature: ENDOSOMAL ESCAPE, i.e. your second question: “where are the smartflares?”. Ideally, the aim here should be to provide a higher boundary estimate for the proportion of nanoparticles which might be outside of vesicular compartments. We will need some help of Joan and Marie for these experiments. We have quite a lot of data already, but I think that we need to plan three more experiments:
    1-2. Electron microscopy and photothermal imaging (combined with some fluorescent markers) of VEGF smartflares at 24 hours
    3. A live 24 hours experiment (without rinsing). I don’t think that background will be a big issue because the nanoparticles should not be fluorescent prior to coming in the cells.

    I would then discuss MECHANISMS OF FLUORESCENCE INCREASE. The fact that we do not see any major differences between scrambled and target, or between DMOG and minus DMOG, suggest nucleases activity. Here, the micro-injection experiment, especially if it works would be really nice. However, I can see plenty of reasons why it might not work. Cells might stressed to the point where signalling is messed up. Particles could be recaptured internally by clearing mechanisms, etc. Micro-injection is hard to control and slow so N is going to be small. If you wanted to do all the controls & different time points, it would be a lot of work. I would not do this as a top priority. I think it is probably not realistic within our time frame.

    The fluorescence cuvette experiment are important but use too much material ;(. The real time uptake experiment would address that question to some extent. The FLIM too and requires less material. (the test FLIM was promising and could be done both on some pure sample or/and life cells.

    Data analysis. You’re the expert 😉

    Caveolin: looking forward to your post!

    Cheers

    Raphael

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  2. Hi Dave,
    comparing the fluorescence signal from the smartflares with photothermal signal should be very interesting indeed and might go some way to look into the heterogeneity of smartflare fluorescence signal in the cells, i.e. the possible smartflare-positive but perceived VEGF mRNA detection-negative scenario. Me and the microscope both are poised to image whenever you have some samples that you have finished with.
    Cheers,
    Marie

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