How smart are SmartFlares?

This post is co-authored by Raphaël Lévy and Dave Mason.

Note: We contacted Chad Mirkin and EMD Millipore for comments. Chad Mirkin replied but did not allow me to share his comments as he prefers to discuss his work in peer reviewed manuscripts rather than blogs. EMD Millipore has provided a response (reproduced below) and is keen to further engage in the discussion.  They wrote that they “look forward to responding to [the questions you pose at the end of the post] after your blog is posted so other researchers who may have the same questions can follow our discussion online.”


To image proteins in cells, biologists have powerful tools based on the Green Fluorescent Protein (GFP) for which Osamu Shimomura, Martin Chalfie and Roger Y. Tsien  obtained the 2008 Nobel Prize in Chemistry. RNA molecules play crucial roles in cells such as coding, decoding, regulation, and expression of genes, yet they are much more difficult to study. SmartFlares are nanoparticle-based probes for the detection and imaging of RNA in live cells. Could they become the GFP of the RNA world?

Many certainly believe this to be the case. SmartFlare ranked second in TheScientist top ten 2013 innovations, with one of the judges, Kevin Lustig, commenting “These new RNA detection probes can be used to visualize RNA expression in live cells at the single-cell level.”  The following year, SmartFlare won an R&D100 award. The technology comes from Chad Mirkin’s lab at Northwestern University. Chad Mirkin is the winner of numerous prestigious prizes and a science adviser to the President of the United States. The scientific articles introducing the SmartFlare concept (under the name of Nano-Flare) were published in the Journal of the American Chemical Society in 2007, ACS Nano in 2009, etc. In 2013, the SmartFlare technology was licensed to EMD Millipore. Here is one of their promotional video:

For a molecular sensor to work, it needs a detection mechanism. The principle of the SmartFlare is explained from 0:45. A capture oligonucleotide (i.e. DNA) is bound to the gold nanoparticles. A reporter strand is bound to the capture strand. The reporter strand carries a fluorophore but that fluorophore does not emit light because it is too close to the gold (the fluorescence is “quenched”). In the presence of the target RNA, the reporter strand is replaced by the target RNA and therefore released, quenching stops, and fluorescence is detected. The release is shown at 2:05. Simple and convincing. Gold nanoparticles are indeed excellent fluorescence quenchers (we have used this property in a couple of papers).

But, for a molecular sensor to work, it also needs to reach the molecule it is supposed to detect. The SmartFlares are shown at 1:40 entering the cells via endocytosis, a normal mechanism by which the cell engulfs extracellular material by entrapping them into a bag made of cell membrane. Molecules and particles which enter the cell by endocytosis normally remain trapped in this bag. This entrapping is essential to protect us from viruses and bacteria by preventing them from accessing the cell machinery. Here, however, at 1:45 – 1:46, something truly remarkable happens: the endosome (the bag) suddenly fades away leaving the particles free to diffuse in the cell and meet their RNA targets. This is a promotional video so you might say that the demonstration of, and explanation for, this remarkable endosomal escape is to be found in the primary literature but that is not the case.

SmartFlares_scheme

There is an extensive body of literature (not related to SmartFlare) dealing with endosomal escape. Some bacteria (like Listeria which can cause food poisoning) and viruses (like Influenza or HIV) use proteins to destabilise the endosome, escape and cause disease. Other mechanisms involve altering the ion balance in the endosome to pop it like an over-inflated balloon (you can read more about the ‘Proton Sponge Effect’ in this review). The problem is that none of these scenarios are applicable to gold nanoparticles conjugated to oligonucleotides. The problem is compounded by the choice of techniques used to analyse SmartFlare uptake into cells. Most of the published papers (for examples see here, here and here) characterise “uptake” and do so largely via Flow Cytometry or Mass Spectrometry (to measure the gold content of the cells). These papers certainly support NanoFlares being taken up into endosomes, but don’t offer any evidence for endosomal escape. A systematic unbiased electron microscopy study would enable to gather an estimate of how many nanoparticles have escaped the endosomes. Alternatively, fluorescence microscopy can be used to visualise a diffuse (released) instead of punctate (still in endosomes) distribution of intensity. While there are some images of cells having taken up NanoFlares, the sort of resolution required to discern distribution is not afforded by publication-size figures.

Wouldn’t it be nice if we had access to the original data? Researchers are often left squinting at published figures and all too often have to rely on the author’s interpretation of the data. One solution to this problem is to make supporting data available after publication. This is the idea behind the JCB Dataviewer; allowing authors to upload the original data to support papers published in the Journal of Cell Biology. The other option is to make the data available before publication, in what is called Open Research. This has the huge advantage of opening up a discussion about data, its interpretation and meaning before going through the formal peer-review process.

It is this latter technique that we are currently using to share our study of the use of NanoFlares as VEGF RNA reporters in cells. Our Open Science Notebook gives an overview of the experimental design, results and discussion, while our OMERO server is being used to host all of the original data for anyone to access. The project is still in progress, however our main findings so far are that:

In all conditions where fluorescence is seen, the distribution is consistently punctate (see all of the data here ).

So far these findings have left us with several questions, the most interesting of which are:

  1. Why do we see punctate fluorescence with the VEGF SmartFlares? If the SmartFlares are still in endosomes, they shouldn’t be able to interact with mRNA and thus fluorescence should be quenched.
  2. Why do we see signal at all in the scrambled control?
  3. Why do different cells take up varying amounts of SmartFlares? Fluid phase dextran shows approximately equal uptake in all cells.

We’re presently investigating these and other questions. As we find out more, we will continue to post the data and update the blog.


RESPONSE from EMD Millipore:

In their response, EMD Millipore pointed to a number of relevant publications suggesting that we should revise the post after having considered this evidence. We had already seen those references and we have not altered the post, but we reproduce EMD Millipore’s response below:

 Oligo-modified nanoparticle internalization and endosomal release:

·         Oligonucleotide modified nanostructures are taken in through an endocytotic mechanism.  http://www.pnas.org/content/110/19/7625.long

·         These highly anionic structures attract a counterbalancing salt cloud.  http://pubs.acs.org/doi/pdf/10.1021/jp205583j

·         This is thought to be the mechanism of release from endosomes (via osmotic pressure) 

 Observation of punctate fluorescence:

·         At short time points, when these structures are indeed in the endosomes,  or at low detection gains on a microscope (where you are adjusting for the brightest points) the staining appears punctate.  (For example- the light in a room comes from the bulb, which is the brightest, but the room is still lit.  Keeping only the brightest point in a picture would only show you the bulb.)

·         Therefore, with regards to the experiment you’ve already performed, our first suggestion would be to turn up the gain to see cytoplasmic fluorescence.

·         Here for example are some pictures showing nice cytoplasmic stain  http://www.pnas.org/content/109/30/11975/F1.expansion.html

 

Also may be of interest: 

http://www.nature.com/mt/journal/v22/n6/full/mt201430a.html

It may be worth noting some of our more recent examples of SmartFlare in the literature, spanning across cancer and stem cell research on a variety of detection platforms (flow & microscopy).  Here the punctate fluorescence is also observed, but you can also see nice cytoplasmic staining.

·         Seftor et al. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4026856/

 

·         Mehta et al.  http://www.ncbi.nlm.nih.gov/pubmed/24623279
 

THE SCIENCE NEWS CYCLE [2]

The paper (Nano Letters) demonstrated for the first time…

…planar undulations of composite multilink nanowire-based chains (diameter 200 nm) induced by a planar-oscillating magnetic field.

The Very Respectable Scholarly Society Press Release announced that we were moving (swimming even) towards

“…nanorobots that swim through blood to deliver drugs (video)”

Gizmag informed its readers that

“… Nanorobots wade through blood to deliver drugs”

 

The actual article says nothing about “blood” nor “drug”.

THE SCIENCE NEWS CYCLE 1 is here. This could become a regular feature. I am happy to receive suggestions via Twitter, comments below or email.

 

Nanoparticles for Cell Tracking 2015

The UK Regenerative Medicine Platform (UKRMP) Safety Hub is hosting Nanoparticles for Cell Tracking, 21-22 September 2015 at the University of Liverpool (Foresight Centre).

Date: 21-22 September 2015
Start time: 09:30
End time: 17:30
Location: The symposium will take place at the Foresight Centre, University of Liverpool, 1 Brownlow Street, Liverpool L69 3GL.

Open to:

  • Academia
  • Business/industry

Contact: For more information contact Claire Hutchinson,chutch@liv.ac.uk

The UKRMP Safety Hub was established alongside a further four Hubs to address the number of developmental challenges which need to be overcome to successfully translate promising discoveries in the field of regenerative medicine for the benefit of patients. To ensure research connects seamlessly from discovery science through to clinical and commercial application, BBSRC, EPSRC and MRC together formed the UKRMP across UK universities and research institutions. Cell tracking with nanoparticles is a major component of the Safety Hub.

The meeting will include significant time for discussions regarding the achievements, potential, and limitations of nanoparticles for cell tracking and the implications with respect to stem cell tracking in animal models and humans.

Speakers currently confirmed include:

Prof Andrew Tsourkas, Assistant Director of Program in Targeted Therapeutics, University of Pennsylvania; Developing contrast agents for molecular imaging and cell tracking applications
Prof Kostas Kostarelos, Head of Nanomedicine Lab, University of Manchester; Title TBC
Prof Quentin Pankhurst, Director of Institute of Biomedical Engineering, UCL; Biomedical Applications of Magnetic Nanoparticles
Prof Philip Blower, Chair of Imaging Chemistry, Kings College London;Cell tracking with radionuclides, both direct and reporter gene approaches
Prof Jason Davis, University of Oxford; Resonant contrast agents
Dr Tammy Kalber, Centre for Advanced Biomedical Imaging, UCL;Magnetic Targeting and multi-modal imaging
Dr James Dixon, UKRMP Acellular Hub, University of Nottingham;Enhanced delivery of functional molecules into cells
Dr Neill Liptrott, University of Liverpool; Compatibility of nanomaterials with the immunological and haematological systems
Dr Bill Shingleton, GE Healthcare; Title TBC
Dr Gabriela Juarez Martinez, Knowledge Transfer Network; Title TBC

We are accepting abstracts for both oral and poster presentations; if you would like to submit an abstract please follow the instructions on the guidance document and send to chutch@liv.ac.uk no later than30th June 2015.

Those who are successful will be notified and required to register for the meeting. Registration will open shortly after the Abstract Submission deadline.

Extracting diffusion dynamics from the fluctuations in photothermal images

 Dan-z-earth

This is a guest post by Dan Nieves, who was until recently a joint member of Raphael and Dave’s labs. Dan has moved as far as he could go from us: he is now residing in Sydney at the EMBL Australia node for Single Molecule Science at the University of New South Wales.

Today, our paper from my time at Liverpool “Photothermal Raster Image Correlation Spectroscopy (PhRICS) of gold nanoparticles in solution and on live cells was published in the new Royal Society open-access journal, Royal Society Open Science.  This journal is committed to an open peer-review system, thus, the review history and referees comments are viewable alongside the article, and also post publication peer-review in the form of a comments section below the paper is facilitated. Additionally, the data that supports the conclusions of the paper are (and have to be) readily accessible (here at Figshare). This is exciting, as not only are the discussions between authors and referees are available to everyone, but you can also join in the discussion fully after publication with access to the primary data. Therefore, the critical evaluation/re-evaluation of the work is totally encouraged and should never stop!

Our paper describes the development of an extension of photothermal heterodyne imaging; a technique used to detect and image single gold nanoparticles much smaller than the diffraction limit at high signal to noise via scattering induced by laser light absorption (nice explanation here). The extension employs fast raster-scan imaging of the sample in which fluctuations, or “streaks” (top panels, Fig.1), are observed due to the movement of nanoparticles through the detection volume during the scan. From these fluctuations it is possible to extract how fast the nanoparticles are moving from the application of image correlation analyses.  In our particular case, we applied the raster image correlation spectroscopy (RICS) method, developed in the lab of Enrico Gratton (original paper here). Briefly, after acquiring many raster scan images; the images are then spatially correlated with themselves by shifting the image pixel by pixel in all directions (x and y in this case) and calculating the correlation function.  This means repeating fluctuations within the image, i.e., nanoparticle diffusion, will be reflected in the time it takes for the spatial correlation to decay, for example, the spatial correlations for movement of slow moving objects decays quite differently to that of fast moving objects (lower panels, Fig.1). From the spatial correlations the diffusion behavior, such as the diffusion coefficient, can be extracted.  In our case, I applied the analysis to photothermal images of 8.8nm gold nanoparticles diffusing in solutions of different viscosity to verify the PhRICS approach (Fig.1). Here, we were able to extract the diffusion coefficients of the nanoparticles in the different solutions. The advantage of this approach compared to the current photothermal heterodyne techniques for probing diffusion (photothermal tracking and absorption correlation spectroscopy) is that not only can we acquire rapidly information on fast diffusion dynamics, but we can also observe the distribution of nanoparticles over the relatively large area of the image (≈ 40 μm2).

 

Fig.1

Fig.1 – Example of gold nanoparticle diffusion in solutions of different viscosity (top panel) with the corresponding spatial correlations (bottom panel).

We then turned our attention to the use of the technique to observe the diffusion of fibroblast growth factors labelled with gold nanoparticles on live cells. FGFs are involved in a wide range of essential biological processes from the formation of morphogen gradients and signalling to homeostatic control of glucose and phosphate levels.  Here, 8.8 nm gold nanoparticles were used to covalently label single fibroblast growth factor 2 proteins (FGF2-NP: via this method), and then incubated with live rat mammary fibroblast cells (Fig.2).  It was observed previously in our lab that there is significant heterogeneity in FGF2 distribution and diffusion in the pericellular matrix when bound to heparan sulphate. We found the diffusion coefficient of the FGF2-NP could be extracted, and that diffusion measurements were variable depending on the area imaged.  Additionally, it is apparent that the image data contained much more information than we could extract using the simple diffusion model applied.  The observation of the formation and dissolution of intense peaks within the images, added to the 2D-movement of such peaks from image to image (see Movie1), gives more insight into the dynamic long range restructuring of the pericellular matrix of live cells at “short” (μs and ms) and “longer” (secs and mins) timescales.

Clipboard

Fig.2 – Photothermal image of rat mammary fibroblast incubated with 600 pM of FGF2-NP.  Blue boxes indicate the areas where PhRICS imaging was performed on the cell.

Cell_1_600pMFGF2_RICS_5_STACK

Movie1 – PhRICS image series from box 5 in Fig.2

The paper is now available at the Royal Society Open Science , and if your interest has been piqued thus far, I strongly encourage you check the paper out.  Better still would be for you to engage in the post-publication comments section should you have any questions, comments or suggestions.

What’s wrong with that CNRS press release?

Imagine an important public institution, say, for the sake of example, the police.

Imagine that serious and specific accusations of misconduct have been made against a high ranking officer on a whistle-blower website. These have been picked up in the media. Although there is no suggestion that anybody has been physically harmed, those acts, if proved true, may have costed significant amount of public money and may have had severe consequences on the well being of many people and businesses. The media reports are also a concern because of the damage made to the public trust, essential to the police mission.

Imagine then, that the press release announcing the investigation says nothing of the potential consequences of those putative acts, stresses that the serious and specific accusations are in fact only anonymous comments on a website, indicates that the investigation procedure will be completely opaque to public scrutiny with an undefined timeline, and, finally, concludes with an entire paragraph devoted to the glorification of the work of the accused (and indeed highly qualified and otherwise commendable) officer.

This is, of course, science-fiction. The police would not adopt such a course of action because they know full well that this would only disqualify the investigation and do nothing for the prestige of the (maybe wrongly) accused officer.

This is however very close to what two major scientific institutions have just done.

Last week, the CNRS and ETH Zurich published press releases announcing investigations into allegations of scientific misconduct. Retraction Watch, covering these press releases, “found some of the language in the announcements puzzling. Call us old-fashioned, but generally it’s a good idea to actually do an investigation before saying that “the studies’ findings are not in doubt.”

True, especially in the current context. The scientific enterprise is suffering from a reproducibility crisis. One of the drivers of this crisis is the lack of publication of negative results which is itself a combined consequence of the publication system and of the methods of evaluation of researchers based on where they publish rather than what they publish [I got more (serious) congratulations for my April fool spoof paper in Nature Materials than for my PloS One paper published the day after].

Scientific institutions such as the CNRS and ETH Zurich should be leading the way to change those practices. They should not, at the onset of an investigation, rule out that “studies findings” (maybe) based on data manipulation are *not* in doubt. Instead, they should set firm plans to test how much of this body of work is solid and how much is not. Surely damages to human knowledge and the integrity of the scientific record should be major sources of concern, yet they barely feature in the press release. It would seem that the main (and almost exclusive) concern related to accusations of scientific misconduct is the damage done to the accused until proven guilty/innocent. That concern for individuals is warranted. It should not stop to the accused. If the charges are proved correct, then there are probably a number of other individuals, less prominent and well-known, who have directly suffered to different extent and for whom redress is unlikely to ever happen: the reviewers of papers and grants who have wasted their time on “diagram/chart” which had been “manipulated”; the competitors which may not have had access to such impressive data and therefore would have failed with their papers and grant applications; the PhD students who might have spent three years trying to reproduce some of these experiments without success [you would not have heard about this since negative results are not published] and may have left science in disgust at the end of the process, etc.

If you’re interested, see also this conversation about the CNRS press release via Twitter (with critical contributions from @b_abk6 and others).

and the Lab Times editorial with the important open letter by Vicki Vance

and of course, PubPeer

Elena at the MRS

I am catching up after an holiday break. I have not spoken yet with Elena who was at the MRS spring conference in San Francisco, but, thanks to blogging, I can tell she seemed to have had a good experience.

Fellow blogger Mary Nora Dickson enjoyed Elena’s first oral presentation at an international conference:

peptide SAMs on gold NPs

Elena Colangelo spoke today in GG about her work on whether the curvature of gold NPs will affect the conformation of adsorbed proteins. This is an important topic, with wide ranging applications from drug delivery to energy. She found that more highly curved NPs inhibit hydrogen bonding, decreasing the amount of beta sheet secondary structures. This work will help to inform future investigations seeking to modify nanoparticles with functional ligands. Thanks!

Thank you Mary for the report and congratulations to Elena ;)

Elena attended some great talks:

Carlo Montemagno’s talk

What an inspiring talk!

On the last slide of his talk, Michelangelo’s quote: The greatest danger for most of us is not that our aim is too high and we miss it, but that it is too low and we reach it

He gave an overview of the cutting-edge projects (in the general areas of environment, health and energy) going on in his lab, IngenuityLab.

The project that fascinated me the most is the 4D Printer, where the fourth dimension is intended to be the functionality of the complex system built up by single molecules. The general concept is the precise assemble of the functional building blocks found in nature to give new functionalities to the system, where these functionalities are meant to address issues regarding energy, environment and human health.

It may sound too futuristic, but would you ever have imagined having your smartphone, as it looks like today, 10 years ago?

Neelkanth Bardhan’s talk

I had the pleasure to listen to Neelkanth Bardhan’s talk, Gold MRS graduate student awardee, at Symposium GG.

First, I want to say that I found his presentation very clear and easy to follow, nice layout of the slides.

He first went through the motivation of his work: there is the clinical need of safer (compared to X-rays) and less expensive (compared to MRI) detection technologies. He then presented his work aiming to answer this need: developing a biologically-templated nanomolecular probe for high-resolution in vivo sensing and detection. His modular probe is constituted of M13 virus coating single-walled carbon nanotubes (SWNTs). To this construct desired fluorescent dyes and specific targeting ligands can be attached. His results in vivo have shown how this probe is able to target tumours and can be used during real-time surgical intervention.

More details on his work and successful applications of this probe can be found here.