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
 

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.

A good day for science; respect to the Editor…

Earlier, I reported on the publication of our article on the internalisation of peptide-capped nanoparticles in cells. Today, I want to share with you the publication process as it happened at PloS One. The paper was submitted on the 20th of November 2014. The academic editor sent his decision, major revision, along with two referees reports on the 22nd of December, i.e. one month after submission [great turn around time!].

Reviewer 2 was very supportive but reviewer 1 much less so: there appeared to be a real difference of interpretation regarding the impact of cell-penetrating peptides on the intracellular localisation of ingested nanoparticles. The reviewer also requested additional experiments that we could not easily do at this time and that we felt were unnecessary to support our main conclusions. The academic editor himself, Dr Pedro V. Baptista [more on PloS One editorial process here], was author on a paper which in some ways could be seen as conflicting with our results and interpretation. The response to the referees and editors took me a long time to write. It was submitted on the 29th of January. I share it below.

The paper was accepted on the 6th of February. I welcome this decision, not just because our paper gets published -this is of course also great news!-, but because it demonstrates that there is space for open scientific debate in the peer reviewed literature. For this, I am immensely grateful to Dr Baptista.


Response to the referees.

Dear Dr Pedro V. Baptista

On behalf of my co-authors, I would like to thank you for handling our article and to thank the reviewers for their careful reading and for their comments.

Reviewer 2 notes that the context of our ms is the existence of conflicting reports on the effect of TAT and HA2 on intracellular fate of nanoparticles. Indeed, some articles have reported efficient access to the cytosol, while other studies indicate that most particles remain confined in endosomal compartments. Our own experiments are in line with this second group of articles. Reviewer 2 notes that “the study is well designed and executed and the results are interpreted appropriately”. Reviewer 2 supports publication in its current form.

Reviewer 1 has concerns about novelty. Reviewer 1 also suggests that we should add three references. These fall in the first category mentioned above, i.e. articles that support the notion that TAT enables access to the cytosol. It is of course appropriate that we should cite studies from both groups of articles. One of the three, […], was in fact already cited. We have now added the other two, i.e.: […]

Experiments related to this topic have led to many articles in the past 10 years. However, the persistence of conflicting reports and the importance of the topic for many envisioned applications require new insights. This we have provided through the use of imaging modalities that provide information across different scales: electron microscopy measures what occurs to a few nanoparticles in a very small part of the cell; photothermal microscopy measures what happens to the bulk of nanoparticles across a large part of the cell. This combination is thus uniquely able to address, in at least one cell type and a particular formulation of nanoparticle, the fate of TAT-functionalised nanoparticles after they bind to the cell surface.

Below we respond to the detailed queries of reviewer 1 and trust that the manuscript now meets the standards required for publication in PLOS One.

Dr Raphaël Lévy, rapha@liverpool.ac.uk

Detailed response to reviewer 1 queries:
• Novelty. Our article is a significant piece of work that adds useful information towards understanding and clarifying the impact of cell penetrating peptides on intracellular localisation of nanoparticles. The work is novel because it builds on a new imaging methodology that directly images the nanoparticle cores (as opposed to an attached fluorescent molecule) and gives a better overview of an entire cell than just electron microscopy. It is also novel because our peptide self-assembled monolayer approach enables us to do systematic variations of the surface chemistry of the nanoconjugates.
• “To include as a new figure, the extinction spectra of all the nanoconjugates as well as all the scattering spectra […]”. The reviewer is right that extinction spectra are very useful to characterise functionalisation and colloidal stability. We have added the requested figure as Fig. S0. For the conjugates used in Fig. 1, the formation of the self-assembled monolayers results in a minimal shift of the plasmon band of ~1-3 nm. This shift is small compared to the width of the plasmon peak. Because photothermal microscopy relies on absorption at the wavelength of our heating laser which matches the position of the maximal absorbance, differences due to a 1-3 nm plasmon shift are negligible. Interestingly, particles presenting a higher percentage of TAT in their monolayer do show a larger plasmon shift indicative of aggregation. We have modified the paragraph on the formation of the SAMs as follows: “Formation of the monolayer was immediately visible because of the increased colloidal stability and of a small red shift of the nanoparticles plasmon band (Fig. S0). Higher proportions of TAT in the monolayer resulted in nanoparticle aggregation and therefore were not used for further studies (Fig. S0).”

• “To include the images and quantification in Figure 1 with cells only with naked gold nanoparticles and cells only with PEG-gold nanoparticles and compare intensities.” The images and quantification for “cells only” were already included (Fig. 1A and first column of Fig. 1F). We have not included “naked gold”. Instead, as a reference point, we have used PEG-gold particles that have a capping composition made of CALNN and CCALNN-PEG. “naked gold” does not remain naked: non-specific adsorption of proteins, e.g. serum albumin in the cell medium, very rapidly changes the properties of the surface [see for example, Time Evolution of the Nanoparticle Protein Corona, Casals et al., ACS Nano, 2010, 4, pp 3623–3632]. The CALNN and CCALNNPEG composition was optimised, as discussed p 7, line 213-220 and Fig. S2 “Gold nanoparticles uptake decreases with increasing percentages of CCALNN-PEG”. The selected composition leads to minimal uptake as shown in Fig. 1B and the second column in Fig. 1F. From this reference composition, we have made systematic variations where we include defined percentages of the two functional peptides (dHA2 and TAT). For all these conditions, exemplary images are shown in Fig. 1 A-E, additional images are shared via figshare (http://dx.doi.org/10.6084/m9.figshare.1088379) and the quantifications are shown in Fig. 1F.

• “To perform other technique to quantify the gold content […].To include more time points in the TEM studies […]. […] the efficacy results reported by the authors are premature without the additional data described above.” While we agree that the suggested experiments are interesting, they are not necessary to reach the conclusions arrived at in the ms. Those conclusions do not concern “efficacy”, but increased uptake and intracellular localisation. The increase in photothermal signal as well as in the counts of nanoparticles in EM images unambiguously demonstrate increased uptake. The non-homogenous distribution of signal observed in the photothermal images and the electron microscopy analyses unambiguously rule out cytosolic distribution of the nanoparticles. The time point of 3 hours used in our studies is a key point both from the perspective of applications and of cell entry mechanisms. We agree that a systematic analysis as a function of time after uptake would provide further insights into endocytotic mechanisms, but it is outside of the focus of this study. Furthermore, it has been done extensively by cell biologists since the 1950s using a variety of probes. Notably, one of the first applications of gold nanoparticles in biology precisely focused on the mechanisms by which cells probe their external environment (Electron microscopy of HeLa cells after ingestion of colloidal gold, Harford et al., J Biophys Biochem Cytol 1957 3:749-756; reference added into the ms).

The standards in the field have been to publish only one or two representative electron
microscopy images. The photothermal imaging provides a unique means for the reader to understand nanoparticle distribution over biologically representative scales. Importantly, we are sharing here 942 EM images and 37 photothermal images. By publishing all of our data alongside the study [1], we enable other scientists to check and challenge our conclusions and propose alternative hypotheses. PLoS One is a particularly good forum for our article because of its commenting platform where this discussion can continue in the open after the publication of the article.
[1]. DOIs of the data:

10.6084/m9.figshare.1088379, 10.6084/m9.figshare.875584, 10.6084/m9.figshare.875630, 10.6084/m9.figshare.875545, 10.6084/m9.figshare.875477, 10.6084/m9.figshare.874219, 10.6084/m9.figshare.874153, 10.6084/m9.figshare.874033, 10.6084/m9.figshare.873852, 10.6084/m9.figshare.1088399, 10.6084/m9.figshare.1246458, 10.6084/m9.figshare.1246609,
10.6084/m9.figshare.1246622, 10.6084/m9.figshare.1246660, 10.6084/m9.figshare.1246696, 10.6084/m9.figshare.1246707

TAT and HA2 Facilitate Cellular Uptake of Gold Nanoparticles but do not Lead to Cytosolic Localisation

That is the title of a paper now published at PloS One. I am particularly pleased at the publication of this paper and would like to congratulate and thank its authors. They have all moved on from Liverpool. The joint first authors are Umbreen Shaheen and Yann Cesbron (currently in Rennes, France). Third author is Paul Free, now at IMRE in Singapore. The data were previously published at Figshare (links below).

PS: As we are today the second of April and not the first, you can be confident that this is not an April Fool post.

Yann Cesbron

Yann Cesbron

Umbreen Shaheen

Umbreen Shaheen

Paul Free

Paul Free

DOIs of the data:

10.6084/m9.figshare.1088379, 10.6084/m9.figshare.875584, 10.6084/m9.figshare.875630, 10.6084/m9.figshare.875545, 10.6084/m9.figshare.875477, 10.6084/m9.figshare.874219, 10.6084/m9.figshare.874153, 10.6084/m9.figshare.874033, 10.6084/m9.figshare.873852, 10.6084/m9.figshare.1088399, 10.6084/m9.figshare.1246458, 10.6084/m9.figshare.1246609,
10.6084/m9.figshare.1246622, 10.6084/m9.figshare.1246660, 10.6084/m9.figshare.1246696, 10.6084/m9.figshare.1246707

New paper accepted in Nature Materials

Even or odd? The number of functional peptide on a nanoparticle makes a big difference

Even or odd? The number of functional peptide on a nanoparticle makes a big difference

Update: the blog post below was posted on the first of April 2015

There will be a proper press release soon so I can’t tell you too much, but I am really delighted to share with you that we have a paper today accepted in Nature Materials (Impact Factor 36.4). The paper focus on some truly spectacular results that we got last year showing how the Poisson distribution of functional peptides on nanoparticles affect their biological and physico-chemical properties. Specifically, we discovered that particles with an even number of functional peptides behaved very differently from particles with an odd number of peptides per nanoparticles. The effect seems to disappear above ~13 peptides per nanoparticles. The odd nanoparticles, especially 5, 7 and 9,  have remarkable cell-penetrating properties and this could lead to a range of therapeutic applications such as fabricating even better nano-scale Navy Seals to kill the “bad guys”, i.e. cancer cells. Remarkably, while the even gold nanoparticles struggle to get inside mammalian cells, they were able to easily get into plant cells which are normally more difficult to penetrate. This could considerably accelerate the development of nanoparticle-modified trees for street lighting.

A new logo for the CCI…

As most large organisation, Liverpool Centre for Cell Imaging (CCI) has hired top communication consultants and spent tens of thousands of pounds of public money on the design of a new logo…

Liverpool Centre for Cell Imaging (CCI) is asking its users to vote on three proposed new logo designs, all conceived by students/post-doctoral researchers working in the facility.

I am not too sure which one to vote for so I am asking you, my readers for advice. Vote in the poll below and feel free also to add comments and suggestions.

logo