We submitted “Stripy nanoparticles revisited” first to Nature Materials. The reason to choose Nature Materials as a home for our critique is the unwritten rule that journals will only publish criticism of articles if those articles were published in their journal. Nature Materials, as the journal which published the first article in the series as well as two additional key articles (now 4, the latest one being the cover of the November 2012 edition), had a particular duty to host this controversy.
I copy below the letter to Editor which accompanied our initial submission on the 17th of July 2009.
Dear Vincent Dusastre, Editor of Nature Materials
Re: ms entitled “Stripy nanoparticles revisited”
In May 2004, Nature Materials published an article entitled “Spontaneous assembly of subnanometre-ordered domains in the ligand shell of monolayer-protected nanoparticles” by Jackson et al. This was to become the first of a series of articles which now counts 11 research articles by the same group (J Am Chem Soc 2006, 128, 11135; Phys Rev Lett 2007, 99, 226106; Science 2007, 315, 358; Small 2007, 3, 814; Adv Mater 2008, 20, 4243; PNAS 2008, 105, 9886; Chem Comm 2008, 196; J Phys Chem C 2008, 112, 6279; J Am Chem Soc 2008, 130, 798; Nature Materials 2008, 7, 588). All the eleven articles rest on a belief in the existence of “stripy nanoparticles”.
The original article claims that mixed monolayers of thiolated ligands, then mercaptopropionic acid (MPA) and octanethiol (OT), self-segregate and form stripes at the surface of gold nanoparticles. This highly surprising claim is backed up by Scanning Tunneling Microscopy (STM) at the surface of gold nanoparticles with sub-nanometre resolution in air. No other groups had, at that time, or have now, ever imaged with such resolution at the surface of highly curved objects. High resolution in STM is normally obtained on very flat surfaces. Roughness induces feedback perturbations which are likely to be the cause of the observed stripes.
In addition to the above, there are other highly surprising results in the 2004 article which cast doubts on the seriousness of the study. Solubilities of the nanomaterials are reported to be “non-monotonous” as a function of molecular composition but they are in fact qualitative visual observations and are contradicted by the same group in a later publication (PNAS 2008, 105, 9886). One of the major claims of the article is that these “stripy nanoparticles” have a unique ability to prevent non-specific interactions. This claim, made in the abstract and in the ms text, is not backed up by any experimental results; it is only backed up by a cartoon.
In May 2008, Nature Materials published another article of this series. The main claim of that second article is that “stripy nanoparticles”, somewhat magically, go through the membrane of living cells without being captured by the endocytotic pathway. The “stripy” water-soluble nanoparticles used in this study were first described in a Chemical Communication article. Remarkably, the claim that they are stripy is backed up by a single STM image of a single nanoparticle. The same image of the same nanoparticle is used in both the ChemComm and the Nature Materials articles. There are a number of other fundamental problems in the 2008 Nature Materials paper. In particular, observation of the nanoparticles inside the cells relies on a fluorophore attached to the nanoparticle. The possibility that such a modification would alter the molecular structure of the monolayer is not discussed. More importantly, gold nanoparticles are very good fluorescence quenchers so the fluorescence observed may well be due to ligand release from the gold core (ligand exchange), a process which is well documented. Several of the pictures published are saturated in intensity which means that localisation, the key issue in this study, cannot be evaluated.
The impact of this series of paper is significant. Although no group has yet reproduced any of these data, the articles are highly cited and other works in the area of nanoparticle delivery to cells are judged in comparison with this article series. If indeed the claims made in this series stand scrutiny, they represent a major change in our understanding of self-assembly and self-organization, as well as in our understanding of nanomaterials interactions with biomolecules and living systems. If they do not, it is critical for the progress of the field that they are challenged so that discussion and evaluation of novelty can be done on solid bases.
In this Report, we revisit the evidence for the existence of the stripes and for the mechanisms of entry of “stripy” nanoparticles into living cells. Fast Fourier Transform (FFT) is a classical tool for the analysis of periodic structures which is widely applied to scanning probe microscopy images. FFT analysis of the published STM images demonstrates that the stripes are a scanning artefact. We also note that some of the published ensemble measurements (solubility in ethanol) are self-contradictory and we provide new measurements which should be highly sensitive to surface properties and do not point towards the existence of phase transitions in this system.
We examine carefully the colloidal properties and uptake of “water-soluble stripy nanoparticles” in live cells. We use state-of-the-art photothermal microscopy which, contrary to fluorescence, allows the direct observation of the gold core and does not require the insertion of a fluorophore in the self-assembled monolayer. We observe that, in contradiction with earlier reports, the behaviour of the “stripy” nanoparticles is identical to the one of those coated by a homogeneous layer of thiolates.
We believe that it is critical that these results are published with high visibility and that Nature Materials is the most appropriate journal because it published the first paper of the series in 2004 and because it published the more recent article on intracellular delivery in 2008.
The very particular nature of this manuscript raises a number of significant problems for its non-biased evaluation. Potential referees in the field of nanoparticle research are likely to have been involved in the evaluation of one or more of the articles of the series; some potential referees are also co-authors of papers in the series and should therefore be excluded. To ensure the highest degree of scrutiny and avoid the risk of biased evaluation, we suggest that the two main aspects of our study should be evaluated by world experts in the appropriate field as explained below.
Our first key argument is that the FFT analysis of the STM images published in 2004 and 2008 demonstrates that the stripes are a scanning artefact. We would strongly recommend that world-leading STM experts, rather than nanoparticle experts, are asked to evaluate the validity or otherwise of this argument. Potential referees for this task include: [LIST of world expert with reference to relevant work].
Our second key argument concerns intracellular uptake of nanoparticles. We argue that the evidence presented to support the claim that stripy nanoparticles go through the membrane of living cells is not conclusive. We present experimental results, using state-of-the-art microscopy for the direct visualisation of the gold particles, which show that there is no significant difference in the intracellular localisation of nanoparticles covered with a homogenous versus mixed (“stripy”) self-assembled monolayer. We would strongly recommend that world-leading experts in the field of gene/protein delivery and cell imaging are asked to evaluate this section of the ms. Potential referees for this task includes: [LIST of world expert with reference to relevant work]
I have not been in contact with any of the above referees. They have not seen the submitted manuscript. I have no collaboration, direct or indirect, with any of them.
An extraordinary aspect of this case is the fact that subsequent papers of the series have been accepted in a range of different journals. This should not be taken as an indication of the validity of the original claims. Referees have rarely the time to evaluate prior publications and are likely to accept without scrutiny results or concepts which have already been published. The “no non-specific interaction” claim which is not backed by any data in the 2004 paper is a case in point. This claim is repeated in several of the following articles, still without any experimental evidence, but with a reference to the 2004 article.
The possibility of refuting existing data and theories is an important condition of progress of scientific knowledge. The high-impact publication of wrong results can have a real impact on research activities and funding priorities. There is no doubt that the series of papers revisited in this Report contribute to shape the current scientific landscape in this area of science and that their refutation will have a large impact.
I look forward to hearing from you in due course and I hope you will consider favorably this unusual manuscript for publication in Nature Materials.