New publication

The great answer to people saying that #preprints are not peer-reviewed

That perfect title is courtesy of (see tweet below)

On Monday (25/06), we will publish a preprint about the spherical nucleic acid technology. Our paper was prompted by the publication in Nature Biomedical Engineering of “Abnormal scar identification with spherical-nucleic-acid technology” by Yeo et al.

The great answer is… review them! I issued a call to review our preprint before it comes out and I have now sent the article to a number of colleagues across the world. I am very much looking forward to their comments good or bad. The comments will be posted on PubPeer. If you have some time on your hands this Friday or over the weekend to look at the paper, drop me an email and I will also send you a preview copy.

Yeo et al corresponding authors were provided with a copy of our preprint two weeks ago but unfortunately they have not responded. I hope they will post comments on PubPeer. We are planning to subsequently submit a version (hopefully improved thanks to the comments) to Nature Biomedical Engineering. It is however sometime rather difficult to debate the scientific literature through the official channels of traditional journals so this route via preprint will accelerate this important discussion.

 

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Happy holidays, the preprint is out there!

This is a guest post by Marie Held. Credits are given to Andrew Plested for the title/timing of this post (see here).

It had been a long time coming but the preprint of our manuscript Ex vivo live cell tracking in kidney organoids using light sheet fluorescence microscopy is now available on BioRxiv. Together with the preprint, the associated large body of imaging data has been made publicly available in the online Image Data Resource (IDR) repository. Through the power of OMERO you can have a play with the data or even download it and then re-analyse it.

The imaging has been conducted on the Zeiss Z.1 Lightsheet microscope at the Centre for Cell Imaging at the University of Liverpool, whose ever helpful staff enabled us generating lots and lots of image data and providing an efficient infrastructure for data storage as well as support for image and subsequent data analysis. Of course this is only half the story because the imaging would not have been possible without generating the samples first: A big thank you also goes to the students and academic staff in the Institute of Translational Medicine at the University of Liverpool.

In this study we have generated organoids from dissociated and re-aggregated mouse embryonic kidney tissue and imaged them with a light sheet fluorescence microscope. The microscope optically sections the samples, therefore preserving the three-dimensional context of the sample throughout imaging. We have found organotypic kidney structures in the organoids and evidence for the maturation of cells to the point of forming glomeruli, the basic filtration unit of the kidney. A functional assay showed that the developed tubules display secretory function.

Most importantly though, we have also performed live imaging of organoids made from genetically tagged fluorescing cells. The light sheet microscopy setup combines an illumination that is perpendicular to the detection. Therefore, full frame images can be recorded rapidly and only the section of the sample that is recorded is illuminated, thus vastly reducing photobleaching and phototoxic effects that limit long-term live fluorescence imaging in wide field and in particular confocal scanning fluorescence microscopy. We have then tracked the fluorescing cells with the help of an automated algorithm and subsequently analysed the generated tracking data. We have {started to} analyse the tracking data and can now quantitatively compare between experiments.

Yet, there is so much more that can be done with the images and data and we would love to see which ideas and approaches others might have, so please do not be shy to dig in and have a play. Be sure to let us know though.

Featured image caption: Organoid of mouse embryonic kidney cells formed following dissociation and re-aggregation of embryonic kidney rudiments. Yellow: Pax2+ cells indicating the metanephric mesenchyme, a prerequisite for nephron development, Red: Peanut-agglutinin staining basement membranes of the ureteric tree and developed nephrons.

Three little (nano) controversies and their morals

This post is a translation of an article originally published in French in Médecine/Sciences. The Editorial of the same issue (also in French) by Pierre Corvol is entitled Scientific integrity: the need for a systemic approach (open access).  You can download a pdf of my article, or, read at the publisher’s website (subscription). For citation, please refer to the original article as follows:

Trois petites (nano) controverses et leurs morales; Raphaël Lévy; Med Sci (Paris), 33 8-9 (2017) 797-800; Publié en ligne : 18 septembre 2017; DOI: https://doi.org/10.1051/medsci/20173308027

« Selon que vous serez puissant ou misérable, les jugements de cour vous rendront blanc ou noir » [1] [Depending on your social height, The law will see your crime as black—or else as white.] Thus concludes the Fable, by Jean de La Fontaine, The Animals sick of the plague : the donkey, guilty of the theft of a few blades of grass, is condemned to death, whilst the Lion and other powerful animals guilty of much more serious crimes are treated to praise and flattery. It is tempting and comforting to think that scientific judgments are of an altogether different nature. Seen in this light, science would reside outside of power struggles and the few mishaps (mistakes, frauds, conflicts of interest) would be rapidly corrected since the reality of the material world would quickly come back to us through experimental results if we were to try to ignore it for too long. The truth is however very different. A large fraction of published scientific results cannot be reproduced. It is not a few mishaps but structural problems which affect the foundations of the scientific enterprise [2, 3]. Peer evaluations seems to encourage the publication of extraordinary stories in high impact factor journals rather than careful and rigorous experimental studies. Contradictory or “negative” data are rarely published: scientific journals are not really interested, and us, scientists, are not particularly motivated by publicly stating our doubts on the work of colleagues who could be in charge of evaluating our next article or grant application. It is particularly urgent to repair our knowledge production system because science is at the center of numerous challenges critical for the future of human beings and the planet. The (real) problems of reproducibility have already been harnessed by lobbies to attack the credibility of scientists [4]. After the election for president of the largest scientific and military power of the world of a man who denies climate change, is very positive about the use of the atomic bomb, and, more broadly wages an open war against science and truth [5], we have a paramount need for science to be open, robust, capable of defending its independence, integrity and universal values. This seems a distant prospect.

The near absence of critical discussion in the scientific literature in many areas of science could make us forget that controversies are an essential aspect of the quest for knowledge, allowing to identify weak points of experiments and theories, thus enabling to consolidate or invalidate them [6]. They are consubstantial to the scientific practice [7]. The analysis of controversies is also a tool to “symmetrically map” the actors to better understand the roles of individuals and social processes [8]. In this piece, I describe three recent controversies in my area of research: gold nanoparticles applied to biology and medicine. This is no “symmetric map”: I am not a neutral observer but a scientist active, to various degrees, in each of those. I am trying nevertheless to draw some lessons and suggestions to improve the ways we work as scientists.

Stripy Nanoparticles

In 2004, Francesco Stellacci’s group at the Massachusetts Institute of Technology (MIT) published in the prestigious journal Nature Materials an article describing gold nanoparticles covered by a mixture of two molecules that self-assemble to form stripes that are observed by scanning probe microscopy [9]. This article and the numerous other ones that will follow in the same journal and in others just as prestigious such as the Journal of the American Chemical Society [10], Science [11] and Proceedings of the National Academy of Sciences (PNAS) [12], suggest that, thanks to their stripes, these nanoparticles have unique properties in terms of wetting, self-organization, interaction with proteins, penetration in cells, with lots of potential applications for biomolecular sensing, or even drug delivery. These articles certainly contributes to the progress of their authors’ careers, but the stripes are an experimental artefact well known by users of scanning probe microscopy. How to explain then that more than 20 “stripy” articles were published between 2004 and 2012? It is obvious that specialists (and even enlightened amateurs) had identified the problem as early as 2004. Yet, the articles and reviews of that period show no sign of it. One now knows that Predrag Djuranovic has been the first to engage into a scientific investigation aiming at testing, and eventually, contesting, the evidence for the the existence of the stripes. In 2005, this rigorous and brave scientist was a student in Francesco Stellacci’s lab. His experimental results and numerical simulation showing how the stripes originate from a poorly adjusted feedback control system were unambiguous but MIT ensured that these results would remain secret [13]. In 2007, I submitted a technical comment responding to the Science article. This first attempt, limited in its scope to the Science article itself, was unsuccessful: Science did ask Francesco Stellacci to respond but then decided not to publish the exchange of views [14]. In 2008, a new article from the MIT group, again in Nature Materials, report that, thanks to their stripes, these nanoparticles can cross the cell membrane and directly access the cytosol [15]. This is accompanied by a commentary entitled “Particles slip cell security” [16]. After discussions with several of my students, we decide to propose a more exhaustive answer. A few months later, the article “Stripy Nanoparticles Revisited” is ready. It includes a new analysis of the stripy images concluding that the stripes are a scanning artefact as well as a critical discussion of the physicochemical and biological properties which, together with experimental results, contradict the claim of direct access to the interior of cells. The article is first submitted to Nature Materials (rejected), then NanoLetters (rejected), and, finally, Small… where it is published after an editorial process that lasted three years [17-19]. The publication of our article, in November 2012, does not end the controversy. Instead, it expands in the scientific literature (a little) and it also takes new forms (in particular on my blog and others [20-23]). Problems with the reuse of images in different publications emerge and eventually lead to two corrections ([12] and [15]). After a number of requests, Philip Moriarty and Julian Stirling (School of Physics and Astronomy, university of Nottingham, UK) are given access to the original data of the 2004 article. They demonstrate, among other things, that the stripes are present in the entire image, i.e. even between the gold nanoparticles [24], a conclusion still rejected by Francesco Stellacci [25].

Homeopathic nanoparticles

The laboratory of Molly Stevens at Imperial College is one of the most prestigious in the field of biomaterials. In 2012, two articles from the group relate the particularly interesting properties of nanoparticles for diagnostic applications. The first one, published in Nature Materials, reports a phenomenon which is entirely extraordinary in which the signal detected increases when the concentration of molecules to detect decreases (“inverse sensitivity”) [26]. Even more incredible, this phenomenon extends to the point where there is less than a molecule of enzyme, on average, in the volume under study. The second article published in Nature Nanotechnology goes further : no need for instruments, the detection of concentration of analytes in the same range is achieved thanks to a colour change visible with the naked eye [27]. Detailed critiques of these articles are available on the website PubPeer [28, 29] as well as in a preprint authored by Boris Barbour; the objections are both simple and profound but the authors have chosen not to respond. One can note that the Avogadro number includes lots of zeros (630 000 000 000 000 000 000 000) and that the detection of a macroscopic change of property due to the presence of a single molecule is therefore an achievement that requires extremely solid proofs. One of the posts on PubPeer indicates that someone contacted the Editor of Nature Nanotechnology in January 2013, but, four years later, no doubts are expressed on the journal website, in the traditional scientific literature nor in the newspapers that had covered this story (e.g. Le Monde and the Daily Mail) when it was initially published [30, 31].

Spherical Nucleic Acids

The laboratory of Chad Mirkin at Northwestern University (USA) is one of the most prestigious in the field of nanosciences applied to biology and medicine. One major theme of their research are the Spherical Nucleic Acids (SNAs), a term introduced by Mirkin to describe gold nanoparticles functionalised with DNA (or RNA) strands. These SNAs are supposed to have properties very different from linear DNA [32]. In particular, they can access the cytosol of live cells, where they could detect and regulate, the presence and quantity of mRNAs. One could ask why this solution did not appear during evolution : to access the cell machinery, viruses and bacteria would have only needed to package themselves within their genetic materials. The first articles (in Science [33], the Journal of the Americal Chemical Society [34], NanoLetters [35], ACS Nano [36]) proposing this surprising theory do no mention the mechanism of the SNAs into cells whatsover. The following one, e.g. [37], propose that the particles enter by endocytosis, but do not explain the mechanism by which the SNAs would escape endosomes. After several dozens of articles on this topic, the proportion of particles reaching the cytosol is still to be measured and reported (in spite of the fact that gold nanoparticles have been used since the 1950s to study intracellular trafficking; such a study would not be difficult). One article from the Mirkin group suggests that SNAs are degraded in the endosomes and that a “small unquantifiable portion escapes […]” [38]. Nevertheless, the particles are now commercially available under the name SmartFlares (Merck Millipore) to detect RNA inside cells. We have studied the entry of nanoparticles in cells and their ability to detect RNA. Given our difficult experience with the publication of Stripy Nanoparticle Revisited, we decided to adopt a different strategy. The project has been open and we have shared our results in quasi real time on our blog. In contradiction with the descriptions made by Mirkin and by Merck Millipore, we have observed that the SmartFlares were degraded in endosomes and were not able to detect mRNA.  Mirroring the tale of Predrag Djuranovic and the stripy nanoparticles, we were not the first to have doubts about the technology: Luke Armstrong, who had been in charge of developing the SmartFlares at Merck Millipore in California (before leaving the company) had reached the same conclusion [39]. To ensure speedy publication and transparency, we published our article on the (not so prestigious) ScienceOpen platform where peer review occurs after publication [40]. We invited comments by Mirkin to no avail. Another article by the same group in PNAS describe a new version of the SmartFlares [41]. Our analysis of the raw data (obtained after multiple insistent requests) show that the signal comes from endosomes. Our letter submitted to PNAS has been rejected by the editorial board because it “[did] not contribute significantly to the discussion of this paper” [42].

 Morals

Access to raw data is essential and guaranteed by clear rules adopted by Universities, scientific journals and funding agencies. It is therefore generally possible to access data with some efforts. It is obviously preferable to publish data at the same time as the articles. This is already the norm for some categories of results and it should become generalised. Researchers should also adopt the Manifesto for reproducible research [43]. The tools are in place to improve the practice of science.

Evaluations of science and scientists must imperatively be based on a critical analysis of their work and the robustness or their results, not on the prestige of the institutions or journals. This requires a change of mind and a clear commitment from researchers who are in positions of power, i.e. everyone who features on promotion or recruitment committees. To say that an article is good because it has been published in a prestigious journal is a moral and logical error which needs to be challenged.

Institutions and scientific journals are not motivated by the quest for scientific truth. The decisisons taken by MIT (keeping Predrag Djuranovic’s findings secret), by Nature Materials (not publishing the exchange with Francesco Stellacci [14]), and by PNAS (not publishing [42]) have directly impacted progress of knowledge. These institutions have commendable principles but, in practice, they aim first at defending their reputation and finances [44]. The latter objective only partially aligns with scientific progress which requires rapid and open discussion of results and conclusions. The Worldwide Web, invented for the sharing of science, enables this discussion. Researchers therefore should embrace the following tools: 1) Pubpeer to comment on articles; 2) Preprints to publish rapidly, minimise the influence of editors, and, dissociate publication, i.e. sharing of information, from evaluation, i.e. peer review; 3) Social networks, e.g. Twitter and blogs, which constitute an ongoing scientific conference to discuss experiments, results, methods, analyses, and new publications.

Acknowledgements: I thank Marianne Noel (IFRIS) for her critical reading of this piece, and, Marianne Lévy for comments on the grammar and style [French version] very necessary after 14 years in an English-speaking country…

Conflicts of interest: The author declares that he has no conflict of interest related to this article.

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Quantum dots for Immunofluorescence

Guest post by Dave Mason

In modern cell biology and light microscopy, immunofluorescence is a workhorse experiment. The same way antibodies can recognise foreign pathogens in an animal, so the specificity of antibodies can be used to label specific targets within the cell. When antibodies are bound to a fluorophore of your choice, and in combination with light microscopy, this makes for a versatile platform for research and diagnostics.

Most small-dye based fluorophores that are used in combination with antibodies suffer from a limitation; hit them with enough light and you irreversibly damage the fluorochrome, rendering the dye ‘invisible’ or photobleached. This property is the basis of several biophysical techniques such as Fluorescence Recovery After Photobleaching (FRAP) but for routine imaging it is largely an unwanted property.

Over 20 years ago, a new class of fluorescent conjugate was introduced in the form of Quantum Dots (QDots); semiconductor nanocrystals that promised increased brightness, a broad excitation and narrow emission band (good when using multi-channel imaging) and most importantly: no photobleaching. They were hailed as a game changer: “When the methods are worked out, they’ll be used instantly” (ref). With the expectation that they would “…soon be a standard biological tool” (ref).

So what happened? Check the published literature or walk into any imaging lab today and you’ll find antibodies conjugated to all manner of small dyes from FITC and rhodamine to Cyanine and Alexa dyes. Rarely will you find QDot-conjugated antibodies used despite them being commercially available. Why would people shun a technology that seemingly provides so many advantages?

Based on some strange observations, when trying to use QDot-conjugated antibodies, Jen Francis, investigated this phenomenon more closely, systematically labelling different cellular targets with Quantum dots and traditional small molecule dyes.

Francis_et_alFig3_GM

Figure 3 from doi:10.3762/bjnano.8.125 shows Tubulin simultaneously labelled with small fluorescent dye (A) and QDots (B). Overlay shows Qdot in green and A488 in Magenta. See paper for more details. See UPDATE below.

The work published in the Beilstein Journal of Nanotechnology (doi: 10.3762/bjnano.8.125) demonstrates a surprising finding. Some targets in the cell such as tubulin (the ‘gold standard’ for QDot labelling) label just as well with the QDot as with the dye (see above). Others however, including nuclear and some focal adhesion targets would only label with the organic dye.

2190-4286-8-125-graphical-abstract.png

The important question of course is: why the difference in labelling when using Quantum Dots or dyes? This is discussed in more detail in the paper but one explanation the evidence supports is that it is the size of the QDots that hinder their ability to access targets in the nucleus or large protein complexes. This explanation further highlights how little we really know about the 3D structure of protein complexes in the cell and the effect of fixation and permeabilisation upon them. Why for example, can tubulin be labelled with QDots but F-actin cannot, despite them both being abundant filamentous cytosolic structures? At this point we can’t say.

So why is this study important? Publication bias (the preferential publication of ‘positive’ results) has largely hidden the complications of using QDots for immunofluorescence. We and others have spent time and money, trying to optimise and troubleshoot experiments that upon closer study, have no chance of working. We therefore hope that by undertaking and publishing this study, other researchers can be better informed and understand when (or whether) it might be appropriate to use Quantum Dots before embarking on a project.

This paper was published in the Beilstein Journal of Nanotechnology, an Open Access, peer-reviewed journal funded entirely by the Beilstein-Institut.

UPDATE [2017-06-13]: in response to a comment below, I’ve updated the overlay figure to use green/magenta instead of green/red. The original figure can be seen in the paper or here.

How to Characterize Gold Nanoparticles’ Surface?

Guest post by Elena Colangelo

Our Topical Review on the characterization of gold nanoparticles (GNPs) has just been published in the Bionconjugate Chemistry Special Issue “Interfacing Inorganic Nanoparticles with Biology”.

The literature is abounding in works on GNPs for applications in biology, catalysis and sensing, among others. GNPs’ appeal resides in their optical properties, together with the well-developed methods of synthesis available and the possibility of functionalizing their surface with small molecules of interest, which can readily self-assemble on the GNPs’ surface forming a monolayer.

However, allegedly the structure and organization of self-assembled monolayers (SAMs) at the GNPs’ surface are in fact aspects too often neglected [though surely not on this blog; RL]. Such elucidation is challenging experimentally, but it is crucial not only to ensure reproducibility, but also to design nanosystems with defined (bio)physicochemical and structural properties, which could then be envisioned to assemble in more complex systems from a “bottom-up” approach.

Our Topical Review gives an overview of the current knowledge and methods available to characterize the GNPs’ surface with different molecular details.

capture

Cartoon illustrating the different levels of GNPs’ surface characterization discussed in the Topical Review.

First, the experimental methods commonly used to provide the basic characterization of functionalized GNPs, such as identification and quantification of the ligands within the monolayer, are detailed with the aid of some examples.

Second, the experimental methods providing information on the monolayer thickness and compactness are reviewed.

Third, considering that the SAM’s thickness and compactness do not only depend on the amount of ligands within the monolayer, but also on their conformation, the experimental methods that can provide such insights are recapitulated. However, we also stressed on the limitations intrinsic to these methods and on the challenges associated to the determination of the structure of SAMs on GNPs.

Fourth, we summarized some of the approaches used to give insights into the organization of different ligands within a SAM. Indeed, mixed SAMs on GNPs are useful since they can impart to the nanoparticles different functionalities and offer a way to tune their stability.

Fifth, highlighting again the limited insights into the SAM’s structure and organization that can be gathered with experimental techniques, we detailed some examples where a combination of experimental and computational approaches was able to provide a compelling description of the system and to assess molecular details that could not have been revealed experimentally.

Overall, this Topical Review gives emphasis on the importance of GNPs’ surface characterization and on fact that even though a number of experimental techniques are available, they are intrinsically limited and they cannot provide a fully detailed picture. Hence, it is advantageous to combine experimental and theoretical approaches to design nanoparticles with desired (bio)physicochemical properties [such as, e.g., our paper under review, currently available as a preprint; RL].

How to Elucidate the Structure of Peptide Monolayers on Gold Nanoparticles?

I have recently submitted my PhD thesis and we have now pre-printed on bioRxiv the work constituting its major chapter. Together with the pre-print, the data have been made publicly available in an online repository of the University of Liverpool. Well isn’t it perfect timing that this week is open access week? 😉

This work has been conducted nearly entirely during the 2 years of my PhD spent at the A*STAR Institute of Materials Research and Engineering (IMRE) and at the A*STAR Institute of High Performance Computing (IHPC) in Singapore.

In this study, peptide-capped gold nanoparticles are considered, which offer the possibility of combining the optical properties of the gold core and the biochemical properties of the peptides.

In the past, short peptides have been specifically designed to form self-assembled monolayers on gold nanoparticles. Thus, such approach was described as constituting a potential route towards the preparation of protein-like nanosystems. In other words, peptide-capped gold nanoparticles can be depicted as building-blocks which could potentially be assembled to form artificial protein-like objects using a “bottom-up” approach.

However, the structural characterization of the peptide monolayer at the gold nanoparticles’ surface, essential to envision the design of building-blocks with well-defined secondary structure motifs and properties, is poorly investigated and remains challenging to assess experimentally.

In the pre-printed manuscript, we present a molecular dynamics computational model for peptide-capped gold nanoparticles, which was developed using systems characterized by mean of IR spectroscopy as a benchmark. In particular, we investigated the effect of the peptide capping density and the gold nanoparticle size on the structure of self-assembled monolayers constituted of either CALNN or CFGAILSS peptide.

The computational results were found not only to well-reproduce the experimental ones, but also to provide insights at the molecular level into the monolayer’s structure and organization, e.g., the peptides’ arrangement within secondary structure domains on the gold nanoparticle, which could not have been assessed with experimental techniques.

Overall, we believe that the proposed computational model will not only be used to predict the structure of peptide monolayers on gold nanoparticles, thus helping in the design of bio-nanomaterials with well-defined structural properties, but will also be combined to experimental findings, in order to obtain a compelling understanding of the monolayer’s structure and organization.

In this sense, we would like to stress that, in order to improve data reproducibility, enable further analysis and the use of the proposed computational model for peptide-capped gold nanoparticles, we are making the data and the custom-written software to assemble and analyse the systems publicly available.

picture1

Snapshots of the final structure of the simulated 5 (left) and 10 (right) nm CFGAILSS-capped gold nanoparticle, illustrating different content and organization of secondary structure motifs.

Gold nanorods to shine light on the fate of implanted stem cells

people_JC

Joan Comenge

This is a guest post by Joan Comenge

Our work regarding the use of gold nanorods as contrast agents for photoacoustic tracking of stem cells has been just published (or here*). You can find all the technical details of the work there, so I will try to explain here the work for the readers who are not very familiar with our field.

It is important to have the appropriate tools to evaluate safety and efficacy of regenerative medicine therapies in preclinical models before they can be translated to the clinics. This is why there is an interest in developing new imaging technologies that enable real time cell tracking with improved sensitivity and/or resolution. This work is our contribution to this field.

To distinguish therapeutic cells from the patient’s own cells (or here from the mouse’s own cell),  the therapeutic cells have to be labelled before they are implanted. It is well known, that biological tissue is more transparent to some regions of the light spectrum than others. This fact is very easy to try at home (or at your favourite club): if you put your hand under a green light, no light will go through it, whilst doing the same under a red light the result will be very different. That means that red light is less absorbed by our body. Near infrared light is even less absorbed and this is why this region of the spectrum is ideal for in vivo imaging. Therefore, we made our cells to absorb strongly in the near infrared so we can easily distinguish them.

Gold nanoparticles of different sizes and shapes (synthesis and picture by Joan Comenge).

Gold nanoparticles of different sizes and shapes (synthesis and picture by Joan Comenge).

To do this, we labelled cells with gold nanoparticles. Interestingly, the way gold nanoparticles interact with light depends on how their electrons oscillate. That means that size and shape of the nanoparticles determine their optical properties, and this is one of the reasons why we love to make different shapes of nanoparticles. In particular, gold nanorods strongly absorb in the near infrared and they are ideal contrast agents for in vivo imaging.

Capture

Figure reproduced from: The production of sound by radiant energy; Science 28 May 1881; DOI: 10.1126/science.os-2.49.242

We have now cells that interact with light in a different way than the tissue. The problem is that light is scattered by tissue, so resolution is rapidly lost as soon as you try to image depths beyond 1 mm. Obviously, this is not the best for in vivo imaging. Luckily for us, Alexander Graham Bell realised 130 years ago that matter emits sounds when is irradiated by a pulsed light. This is known as the photoacoustic effect and it has been exploited recently for bioimaging. Photoacoustic imaging combines the advantages of optical imaging (sensitivity, real-time acquisition, molecular imaging) and the good resolution of ultrasound imaging because ultrasounds (or phonons), contrarily to photons, are not scattered by biological tissue.
GNR-35.2Si3 in cells_16

Silica-coated gold nanorods inside cells

To optimise the performance of our gold nanorods, we coated them with silica. Silica is glass and therefore it protects the gold core without interfering with its optical properties. This protection is required to maintain gold nanorods isolated inside cells since nanorods are entrapped in intracellular vesicles, where they are very packed. The absence of a protective coating ultimately would result in a broader and less intense absorbance band, which would be translated to a less intense photoacoustic signal and consequently a lower sensitivity in cell detection. This of special importance in our system, a photoacoustic imaging system developed by iThera Medical which uses a  multiwavelength excitation to later deconvolute the spectral information of the image to find your components of interest. Thus, narrow absorption bands helps to improve the detection sensitivity even further. With this we demonstrated that we were able to monitor a few thousand nanorods labelled-cells with a very good 3D spatial resolution for 15 days. This allowed for example to see how a cell cluster changed with time, see how it grows or which regions of the cell cluster shows the highest cell density. In addition, this work opens the door to new opportunities such as  multilabelling using gold nanorods of different sizes and consequently different optical properties to observe simultaneously different type of cells. We also believe that not only stem cell therapies, but also other fields that are interested in monitoring cells such as cancer biology or immunology can benefit from the advances described in our work.

You can find the original publication here (or here*).
All the datasets are available via Figshare.

This work was supported by the UK Regenerative Medicine Platform Safety and efficacy, focusing on imaging technologies. Joan Comenge was funded by the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme. The in vivo imaging was done in the Centre for Preclinical Imaging, the Electron Microscopy in the Biomedical EM unit and the Optical Microscopy in the Centre for Cell Imaging.

* the alternative link is to 50 free e-prints; the link will be removed once the paper is fully open access (in a couple of days).

day3_200k

Cluster of gold nanorod-labelled cells imaged by photoacoustic imaging three days after implantation in mice.