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Do nanoparticles deliver? Merck’s SmartFlares and other controversies

Raphaël Lévy:

Leonid Schneider’s article starts with a summary of the stripy controversy and then moves on to the SmartFlare. Of particular interest is the quote from Luke Armstrong, formerly at EMD Millipore, which demonstrates that the company ought to be well aware that the probes detect nucleases rather than mRNAs. This begs the question of why they are still selling and advertising this product. Unfortunately, they did not provide a statement to Leonid. [Picture above is from Leonid’s post]

Originally posted on For Better Science:

A large body of scientific nanotechnology literature is dedicated to the biomedical aspect of nanoparticle delivery into cells and tissues. The functionalization of the nanoparticle surface is designed to insure their specificity at targeting only a certain type of cells, such as cancers cells. Other technological approaches aim at the cargo design, in order to ensure the targeted release of various biologically active agents: small pharmacological substances, peptides or entire enzymes, or nucleotides such as regulatory small RNAs or even genes. There is however a main limitation to this approach: though cells do readily take up nanoparticles through specific membrane-bound receptor interaction (endocytosis) or randomly (pinocytosis), these nanoparticles hardly ever truly reach the inside of the cell, namely its nucleocytoplasmic space. Solid nanoparticles are namely continuously surrounded by the very same membrane barrier they first interacted with when entering the cell. These outer-cell membrane compartments mature into endosomal and then…

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New data on SmartFlare – do they detect mRNA?

Raphaël Lévy:

Thank you Gal for providing the first peer review of this paper. As mentioned in my post, this is a scientific investigation as well as an experiment in new ways of doing, sharing and evaluating science. I am very grateful for your contribution, coming just 24 hours after initial publication, as it contributes to the success of the latter experiment as well as provides opportunities to improve our paper. We will of course respond to your detailed comments soon!

Originally posted on greenfluorescentblog:

Almost exactly a year ago, I wrote a post regarding my concerns with SmartFlare, supposedly a novel method for live imaging of RNA in cells.

In a nutshell, SmartFlare are gold nanoparticles covered in oligos specific to a certain mRNA of interest. Supposedly, cells internalize these particles and, once the mRNA hybridize to the oligo, a complementary fluorecently labeled oligo is being unquenchhed and “flares”, indicating the present of said mRNA.

You can read about my concerns in that older post, but apparently I wasn’t the only one concerned about their validity.

Raphael Levy from U. of Liverpool (UK) was concerned as well. He endeavored into an open science project to try and answer his concerns (which is why I allow myself to openly review his paper).

Raphael’s concerns were that these gold nanoparticles are maintained in endosomes and do not reach the “cytoplasm” where mRNAs reside. Since he…

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The Spherical Nucleic Acids mRNA Detection Paradox

We are publishing today “The Spherical Nucleic Acids mRNA Detection Paradox“, the outcome of an open science project which started with an Hns student last year. In the last 12 months, we have reported in quasi real-time our experiments, protocols and analyses in an open science notebook and shared the data on our repository. The data are also stored at FigShare (e.g. Electron Microscopy results).

In addition to being exciting scientifically (he says!), this has been an experiment in how do science in the open using the tools of the 21st century to share information and solicit feedback. It is therefore fitting to publish it on a platform that challenges conventional modes of peer review.

We have chosen ScienceOpen where publication happens immediately (a couple of hours from submission to publication), followed by open peer review. In the coming weeks and months, I hope that many scientists will provide their expert evaluation of our article. In particular, Chad Mirkin will be invited to provide a review.

This article is important to all scientists who are using nanoparticles for imaging and sensing inside living cells. It should also be particularly relevant to past, current and prospective customers of the SmartFlares. Here is the abstract:

From the 1950s onwards, our understanding of the formation and intracellular trafficking of membrane vesicles was informed by experiments in which cells were exposed to gold nanoparticles and their uptake and localisation, studied by electron microscopy.  In the last decade, building on progress in the synthesis of gold nanoparticles and their controlled functionalisation with a large variety of biomolecules (DNA, peptides, polysaccharides), new applications have been proposed, including the imaging and sensing of intracellular events. Yet, as already demonstrated in the 1950s, uptake of nanoparticles results in confinement within an intracellular vesicle which in principle should preclude sensing of cytosolic events. To study this apparent paradox, we focus on a commercially available nanoparticle probe that detects mRNA through the release of a fluorescently-labelled oligonucleotide (unquenching the fluorescence) in the presence of the target mRNA. Using electron, fluorescence and photothermal microscopy, we show that the probes remain in endocytic compartments and that they do not report on mRNA level. We suggest that the validation of any nanoparticle-based probes for intracellular sensing should include a quantitative and thorough demonstration that the probes can reach the cytosolic compartment.

The paper will be typeset in the next few days and open peer review will be open from that point. Comments are already possible. Thank you to Dave Mason, Gemma Carolan, Joan Comenge and Marie Held for their contributions to this work.

Towards the end of the stripy controversy?

Last week saw the publication in PloS One of Quy Khac Ong and Francesco Stellacci’s response to Stirling et al “Critical Assessment of the Evidence for Striped Nanoparticles” published a year earlier (November 2014, I am one of the co-authors).

The controversy had started with our publication of Stripy Nanoparticles Revisited after a three year editorial process (2009-2012) and was followed by a large number of events at this blog, on PubPeer and a few other places.

Here is a short statement in response to Ong and Stellacci. Since theirs  was a response to Stirling et al, Julian Stirling was invited to referee their submission (report).

We are pleased that Ong and Stellacci have responded to our paper, Critical assessment of the evidence for striped nanoparticles, PLoS ONE 9 e108482 (2014). Each of their rebuttals of our critique has, however, already been addressed quite some time ago either in our original paper, in the extensive PubPeer threads associated with that paper (and its preprint arXiv version), and/or in a variety of blog posts. Indeed, arguably the strongest evidence against the claim that highly ordered stripes form in the ligand shell of suitably-functionalised nanoparticles comes from Stellacci and co-authors’ own recent work, published shortly after we submitted our PLOS ONE critique. This short and simple document compares the images acquired from ostensibly striped nanoparticles with control particles where, for the latter (and as claimed throughout the work of Stellacci et al.), stripes should not be present. We leave it to the reader to draw their own conclusions. At this point, we believe that little is to be gained from continuing our debate with Stellacci et al. We remain firmly of the opinion that the experimental data to date show no evidence for formation of the “highly ordered” striped morphology that has been claimed throughout the work of Stellacci and co-workers, and, for the reasons we have detailed at considerable length previously, do not find the counter-claims in Ong and Stellacci in any way compelling. We have therefore clearly reached an impasse. It is thus now up to the nanoscience community to come to its own judgement regarding the viability of the striped nanoparticle hypothesis. As such, we would very much welcome STM studies from independent groups not associated with any of the research teams involved in the controversy to date. For completeness, we append below the referee reports which JS submitted on Ong and Stellacci’s manuscript.

Julian Stirling, Raphaël Lévy, and Philip Moriarty November 16 2015



PNAS: “your letter does not contribute significantly to the discussion of this paper”

Thanks to BiorXiv, you can read, comment upon, and cite, a letter which, according to PNAS editorial board, does not contribute significantly to the discussion of

William E. Briley, Madison H. Bondy, Pratik S. Randeria, Torin J. Dupper, Chad A. Mirkin, Proceedings of the National Academy of Sciences (2015)
Here is the email from Inder Verma, Editor-In-Chief:

November 16, 2015

Title: “Sticky-flares: real-time tracking of mRNAs… or of endosomes?”
Tracking #: 2015-20468
Authors: Mason and Levy

Dear Dr. Levy,

I regret to inform you that the PNAS Editorial Board has declined to publish your Letter to the Editor. After careful consideration, the Board has decided that your letter does not contribute significantly to the discussion of this paper.

Thank you for submitting your comments to PNAS.

Sincerely yours,
Inder Verma

 You can also check the comments at PubPeer.

A postcard from Israel

Originally published on the University of Liverpool news website.

“I sat next to the taxi driver. We started talking. The ride from my hotel on Ben Yehuda Street in down town Tel Aviv to the Bar Ilan campus takes about 45 minutes. ‘Liverpool’, the taxi driver said, ‘I was there several times in the 1950s; I was a sailor.’

I was sufficiently intrigued to ask him his age. ’80 in six months,’ was his reply. He, who was older than his country, had no shortage of stories and the 45 minutes went quickly. When the Suez crisis broke in 1956, he was in Odessa, Soviet Union, on a tanker ship supposed to bring coal to Israel. I also learnt that Russia is 800 times larger than Israel.

I had spent the previous three days at Tel Aviv University, closer to the city centre, starting the week (on Sunday, of course) by giving a seminar in the Centre for Nanoscience and Nanotechnology. In Bar Ilan, I visited and gave a seminar at BINA, the Institute for Nanotechnology and Advanced Materials. The aim of my 10 days visit, funded by the British Council, is to develop scientific collaborations with Israeli colleagues and so, beyond taxi drivers, I met biologists, physicists and chemists working on areas more or less related to my own research interests, i.e. the preparation of nanoparticles and their applications in biology, especially for imaging of biomolecules and tracking of cells.

The discussions, prompted by my somewhat provocative, or ‘iconoclastic’ as one distinguished Israeli colleague put it, seminar were not limited to nanoscience but extended to the reproducibility crisis, changes in the scientific publishing landscape, and the opportunities that the internet offers to improve the way we do science by increasing the sharing of ideas (including critiques) and the sharing of data.

The tree of knowledge and electronics, and, Ouri’s corner by Asaf Lifshitz.

The tree of knowledge and electronics, and, Ouri’s corner (detail), by Asaf Lifshitz. Picture by Raphaël Lévy.

This week, I complete my journey by visiting a third major Israeli institution, and giving two more seminars, at the Technion, situated in Haifa, also on the coast, an hour by train North of Tel Aviv. The stunning campus is decorated by a number of sculptures. The first seminar takes place in the Institute of Biomedical Engineering and the second in Chemical Engineering.

During these ten days I will have met over 50 scientists at different stages of their careers and I hope that there will be some lasting links; maybe one of the younger ones will join my lab in Liverpool, or, an international collaborative project will take shape with more established colleagues? Whatever happens, if I go again, I’ll send you another postcard from Israel.”