More hype than hope? #Biomaterials16

Congratulations to the organisers of the World Biomaterials Congress for having a high profile debate on the following proposition:

Nanotechnology is more hype than hope

I wish I could have attended as it is a topic I have given some thought… Thankfully, one of the attendees, Professor Laura Poole-Warren has done some live tweeting from the floor. So here is a storify.

A $58M question

Nanosphere, one of the three companies founded by Chad Mirkin, has been bought today for $58M by Luminex. Is this another triumph of the Spherical Nucleic Acids?

Nanosphere was founded in 1999 by Dr. Robert Letsinger and Dr. Chad Mirkin and went public in 2007 (NASDAQ: NSPH). In 2004, MIT technology reports “a powerful but cheap nanotech tool available this year could test for everything from genetic diseases to heart-attack signs.”

Mirkin says that the Nanosphere technology is orders of magnitude more sensitive than other detection techniques, as well as fast and accurate. What’s more, the technology detects DNA or proteins directly, does not require expensive and time-consuming preparation of blood samples, and can test for multiple targets at once. “It will completely change the way the world looks at diagnostics, he says. “I’m very confident that we’re going to see a lot of new diagnostic tools come out of this.”

However, 12 years after this statement and 17 years since foundation, the company is still to make a profit. It has been able to raise and spend huge amounts of money. Not being a financial expert myself, I can’t quite work out the total, but it is probably close to ten times the value it has been sold today. An article last year, entitled “Things are not well at Nanosphere” reported that the company had “burned through $412.5 million since inception”.

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The difficulties of the company, visible for all for example in the evolution of the share prices since 2007 (above), have not led to any nuance in the enthusiastic celebration of Mirkin as a genius entrepreneur leading the way in the translation of nanotechnology into healthcare. Mirkin also benefitted directly through consultancy fees from 2005 to 2013 ($100k per year 2008-2013) and a 2010 news article reports that “while profits have been elusive, Nanosphere has paid off for Mr. Mirkin, who owned 840,000 shares as of this spring, or $2.5 million worth, although the stock has sagged lately…“.

Future will tell how much an important contribution to diagnostic the Spherical Nucleic probes of Nanosphere (now Luminex) will make.

At least, for the moment, it is a better outcome than another Mirkin-founded company, NanoInk.

Update: see reporting by John Pletz from Chicago Business

 

 

The bust of Nelson Mandela and the Professor

nelson-mandela-sculpture-june-20133

Bust of Mandela commissioned by Ken Livingstone in the 1980’s

Foreword/disclaimer: as for all posts here (except when noted otherwise), these are my words and I take full responsibility/credit for them. This post is a bit different from most as it is not about science nor the fabric of science. 

The past week has seen antisemitism making the headlines and the former Mayor of London Ken Livingstone suspended from the Labour party. Here is an anecdote which illustrates why it is so difficult for the left to understand and fight this pervasive prejudice.

The Professor in my anecdote is David Colquhoun, a hero of mine as an advocate for open science, defender of the NHS  and indefatigable fighter against quackery and p-hacking. As many, he tweeted about the antisemitism row (he tweets quite a lot; the few tweets I quote here do not claim to be representative). Here is an early one that caught my attention (and gave this post its title).

What strikes me is the sheer irrationality of the comment. Somehow Ken’s support for Mandela in the 1980’s is seen as relevant to evaluate whether his comments last week were antisemitic. The Professor does not think Ken could be racist. It is simply unimaginable: he is one of the good guys who supported Mandela. Unfortunately, it is impossible to confront that one can not imagine.

In another thread, the Professor asksGenuine Q. Is it “anti-semitic” to deplore treatment of Mordechai Vanunu? Or deplore not signing nuclear non-proliferation?“. The answer is obviously no… and no one had suggested this (and possibly no one has ever suggested this ever). A classical example of the Livingstone formulation, a displacement from a concern about antisemitism to one about “silencing criticism of Israel”. Since the Professor seemed to have a genuine desire to find ways to express his views while avoiding antisemitism, I suggested this piece and noted that:

I suppose my tweet was slightly clumsy: the Professor thought I had accused him of antisemitism. Now, if I am accused of prejudice, any form of prejudice, my reaction will be to seek to understand the charge so that I can learn what I could have done better, and, if necessary, apologize or dispute the charge. Well, that is not how the Professor reacted. Instead, he quoted my tweet to his 12.5K followers:

Antisemitism is not a prejudice, it is an insult. I clarified the misunderstanding (and the Professor accepted the clarification). In the following few days, the Professor would go on to quote various (thoroughly unrepresentative) Jewish voices that would reassure him that no, there really isn’t a problem with antisemitism in the left, and, the Tories are much worse anyway, etc. And then this retweet; a good old conspiracy: the “antisemitism” is within brackets and the story has been “concocted”.

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Some resources:

 

Time-resolved Microscopy and Correlation Spectroscopy

This is a guest post from Jennifer Francis, PhD Student in the group. When group members go to conferences or courses, they have to write a travel blog post.

I recently attended the 8th European Short Course on “Time-resolved Microscopy and Correlation Spectroscopy” at PicoQuant headquarters in Aldershof-Berlin; specifically to learn about the principles and application of FRET, FLIM, and FCS to the Life Sciences. A day prior to this microscopy course, I also attended the 14th SymPhoTime Training Day. As well as discussing problems and applications of the SymPhoTime64 software with other users, I also had the opportunity to speak with a programmer of the software, who demonstrated many new features and assisted with analysis of my own FCS measurements at a computer station. My mornings generally started with passing an incredible sculpture of two heads, before arriving at the Max Born Institute for lectures delivered by scientists in the field of time-resolved microscopy, including Professor Jörg Enderlein.

two heads

Landmark of Berlin-Aldershof: Kopfbewegung – heads, shifting.

 

After a Flammkuchen, I participated in afternoon practical microscopy sessions, where I got the chance to experience the commercially available super-resolution microscope: MicroTime 200 STED, which was awesome. Not only did we see this cutting-edge instrument in action, resolving structures below the diffraction limit, but we also had a peek inside the operating laser boxes! Whilst at the STED station, members of GATTAquant informed us about nanorulers, which are new standards for super-resolution microscopy. Another highlight of this workshop was the demonstration of the Zeiss LSM 880 with Airyscan, which boasts 32 detectors. There were a total of 42 participants on this course, both from academia and industry, including microscope representatives from Olympus, Zeiss, Leica, and Nikon. The consensus take home tip from all participating microscope companies was to always match the refractive index of the objective with that of the sample and to adjust the correction collar to take into account coverslip thickness.

 

SmartFlare Maths

SmartFlare are nanoparticle sensors which are sold by Merck and are supposed to detect mRNA inside live cells. The technology has been developed by Chad Mirkin. In his papers, the nanoparticles are called Nano-Flares or Spherical Nucleic Acids. I am saying “supposed to” because the central question of how those sensors could possibly reach the target that they are supposed to detect has not been addressed by Mirkin nor by Merck.

After evaluating the SmartFlare, we published recently our conclusions at ScienceOpen. We ran this research as an open science project, sharing our experimental results, analyses and conclusions in quasi real time using an open science notebook. All of the imaging data can also be consulted via our online Open Microscopy Environment repository.

Gal Haimovich, who reviewed our paper, first on his blog and then at ScienceOpen, suggested we should do some SmartFlare Maths (point 4 of his list of comments). This had been at the back of my mind for some time. There are various ways to look at this problem, but all those I have tried lead to the same conclusion that the protocols, results and conclusion published do not add up. Here is what I believe the simplest way to think of the SmartFlare Maths problem. As usual, comments and corrections would be very much appreciated.

Estimation of the number of SmartFlares per cell

SF-figure adapted from Giljohann

Adapted from Giljohan et al, Figure 1b

Estimate 1. SmartFlares are added to cells at a final concentration of 0.1 nM (following Merck’s protocol). For 400,000 cells and 20 μL (following Merck’s protocol), this would result in 150,000 SmartFlares per cell, assuming that all nanoparticles are uptaken.

 

Estimate 2. Giljohann et al  (Mirkin’s group) published a quantitative study of uptake of SmartFlares in various cell lines in 2007. From their Figure 1b, we can see that in the lower concentration range tested, there is a linear correlation between SmartFlare concentration in the medium and number of particles per cell. For cells exposed to a medium concentration of 0.1 nM, this would result in an uptake of 75 000 SmartFlares per cell. In the following discussion, we will use this lower estimate. With ~50 oligo probes per SmartFlare, this would give 3,750,000 oligo probes per cell.

Oligo probes per cell versus mRNA per cell

The copy number of any specific mRNA per cell depends on sequence, cell types, signalling events etc, but typically it ranges from a few copies to a few thousands of copies. Our estimate above indicates an excess of oligo probes of at least three orders of magnitude over the most abundant mRNA.

If just 0.1% of these probes would bind their target, it would block 3,750 mRNA resulting in silencing. However, Merck and Mirkin both report that there is no silencing effect in the conditions of these experiments. It follows that more than 99.9% of the SmartFlares do not bind their target mRNA.

Fluorescence background

Seferos

Reproduced from Seferos et al, Figure 1.

Seferos et al (2007, Mirkin’s group) show that in the absence of release of the probe, fluorescence value of ~30% of the total value after release is observed (in ideal test-tube conditions, i.e. in the absence of nucleases). This is presumably due to a non-complete quenching of the fluorescence. For the SmartFlares to work, we would therefore have to detect a variation of less than 0.1% over a background of ~30%.

 

Lab Times: “Flare up over SmartFlares”

Stephen Buckingham interviewed me for Lab Times

On the face of it, Millipore’s SmartFlares are meant to be a tool cell biologists dream of – a way of measuring levels of specific RNA in real time in living cells. But does it really work? Raphaël Lévy and Gal Haimovich are in doubt.

Raphaël Lévy, Senior Lecturer in Biochemistry at the University of Liverpool, UK, was so unconvinced about SmartFlares that he decided to put the technique directly to the test (The Spherical Nucleic Acids mRNA Detection Paradox, Mason et al. ScienceOpen Research). As a result, Lévy has found himself at the centre of a row; not only over whether the technique actually does the job but as to whether it can actually work, at all – even in principle. Lab Times asked Lévy why he is in doubt that SmartFlares really work.

Lab Times:  What’s all the fuss about SmartFlares?

Read it all here (page 50-51).

I can’t resist also quoting this bit of pf the final paragraph…

In interview, Lévy is reasonable and measured in tone. But he is no stranger to controversy and can deliver fierce polemic with style.

If you have not yet, you should also check Leonid Schneider’s earlier and more complete investigation.

Nanoparticles & cell membranes: history of a (science) fiction?

One of the reason scientists, journalists and the general public are excited about nanoparticles is their supposed ability to cross biological barriers, including, the cell membrane. This could do wonders for drug delivery by bringing active molecules to the interior of the cell where they could interact with key components of the cell machinery to restore function or kill cancer cells. On the opposite side of the coin, if nanoparticles can do this, then there are enormous implications in terms of their potential toxicity and it is very urgent to investigate. But is it true? What is the evidence? How did this idea come into the scientific literature in the first place? I have been intrigued by this question for some time. It is the publication of an article about stripy nanoparticles magically crossing the cell membrane that led me to engage in what became the stripy nanoparticles controversy. It is this same vexing question that led me to question Merck/Mirkin claims about smartflare/nanoflare/stickyflare.

In the introduction of our article “The spherical nucleic acids mRNA detection paradox“, we describe the long history of the use of gold nanoparticles (“gold colloids”) in cell biology and conclude that

…, more than five decades of work has clearly established that nanoparticles enter cells by endocytotic mechanisms that result in their entrapment inside intracellular vesicles unless those nanoparticles are biological in nature and have acquired through evolution, advanced molecular tools which enable them to escape.

In the paragraph that followed, we were trying to make the point, in part using citation data of one of these 1950s pioneering articles, that this solid knowledge has been ignored in some of the thousands of recent articles on interactions of nanoparticles with membranes and cells that have appeared in the past 15 years. In his review of the first version of our article, Steve Royle criticises that latter paragraph (in his word, a “very minor” point):

I’m not a big fan of using number of Web of Science search results as an argument (Introduction). The number of papers on Gold Nanoparticles may be increasing since 2007, but then so are the number of papers on anything. It needs to be normalised to be meaningful. It’s also a shame that only 5 papers have cited Harford et al., but it’s an old paper, maybe people are citing reviews that cover this paper instead?

This is a fair point. While normalisation as well as more detailed and systematic searches might shed some light, it is rather difficult to quantify an absence of citation. Instead, I have tried to discover where the idea that nanoparticles can diffuse through membranes comes from. Here are my prime suspects (but I would be more than happy to update this post to better reflect the history of science and ideas so please leave comment, tweet, email), Andre Nel and colleagues, in Science, 3rd of February 2006, “Toxic Potential of Materials at the Nanolevel” :

“ Moreover, some nanoparticles readily travel throughout the body, deposit in target organs, penetrate cell membranes, lodge in mitochondria, and may trigger injurious responses.”

This claim is not supported by a reference, but later in the article Nel et al refer to an earlier paper entitled “Ultrafine particles cross cellular membranes by nonphagocytic mechanisms in lungs and in cultured cells” by Marianne Geiser and colleagues. These two papers, Nel et al, and, Geiser et al, have been cited respectively 5000 times and 850 times according to PubMed.

As early as 2007, Shayla Banerji and Mark Hayes had already challenged this idea of transport of nanoparticles across membranes in an elegant experimental and theoretical study which was a direct response to the two papers cited above “Examination of Nonendocytotic Bulk Transport of Nanoparticles Across Phospholipid Membranes“:

In accordance with these health concerns, Nel et al. have described some phenomena that can only potentiate fear of the negative health risks associated with nanotechnology.

[…]

Non-endocytotic transmembrane transport of large macromolecules is a significant exception to what is presently known about cell membrane permeability. Most early studies show that lipid bilayers are essentially impenetrable by molecules larger than water under physiological conditions: transport of most molecules across cell membranes is specifically cell-mediated by endocytosis.34 Endocytosis, unlike proposed passive, non-endocytotic transport, is an active cell-mediated process by which a substance gains entry into a cell. Specifically, a cell’s plasma membrane continuously invaginates to form vesicles around materials that originated outside the membrane: as the invagination continuously folds inward, the cell membrane constituents simultaneously reorganize in such a way that the material being transported into the cell is completely enclosed in a lipid bilayer, forming an endosome.35,36

[…]

The results suggest that a diffusive process of transport is not likely.

Figure 8 is particularly telling (!).

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The article by Shayla Banerji and Mark Hayes has been cited 44 times.