Critical reading of “Predicting the Time of Entry of Nanoparticles in Lipid Membranes”

Thanks Zeljka for this reading advice: Predicting the Time of Entry of Nanoparticles in Lipid Membranes, by Changyang Liu et al.

So I did. Not the details of the simulations (beyond my expertise) but the introduction and some key aspects of the work, including the choice of nanomaterials and the comparison with experimental results.

I have annotated the article using You can read those annotations here. There are several things in the introduction which are pretty common in the bionano litterature but nevertheless irritating.

So, for example, if you read the title and you are not an expert in this field, you will be thinking that nanoparticles in general enter lipid membranes. But do they? The authors (nearly) clarify in the introduction that in fact they don’t, e.g. “… proteins or nanoparticles (NPs) […] hydrophilicity and large size hamper direct diffusion through the membrane lipid bilayer” and ” In general, cells do not permit access of polar macromolecules to their cytosol, and phospholipid membranes constitute an effective barrier“. But then, they also state the contrary, i.e. that nanoparticle do penetrate cell membranes (science fiction?), e.g. “Smaller nanoparticles can instead cross the membrane by passive transport, that is, by displacing, sometimes irreversibly, the lipids or by diffusing in the hydrophobic region of the membrane and then on the other side“. That extraordinary statement is not supported by references. Which one is true? How do we reconcile these contradictory statements?

The introduction then moves away from the experimental world towards the world of simulation. That is fine (and largely beyond my expertise), but it means the rationale for studying nanoparticles penetration in membranes rests on the juxtaposition of two contradictory statements, one of which is not backed by experimental results.

So the introduction (and title) is about nanoparticles (in general) going through membranes, but the nanoparticles modeled (see below, reproduced from their Figure 8) are three tiny (even for nanoparticles) objects. Their sizes are certainly not typical of contrast agents or drug delivery vehicles discussed in the introduction.

And those nanoparticles have another feature really not typical of nanoparticles for biomedical applications: they are extremely hydrophobic. Their partition coefficient between lipids and water is 100% (table 1 in the paper). This is very far from your typical nanoparticle. And it raises another question related to the experiments presented in the last figure of the article.

UPDATE 24/09 (following Alan’s comment):

Fig 9 (reproduced below) shows the “leaking” of GQDs from giant vesicles (~50-70 micrometers). These are spinning disk confocal images taken with a 60X objectives. So we should see a section through the vesicle (I think it is more likely to be a total intensity projection but they don’t say).

If those “particles” love the membranes so much, they should simply accumulate in membranes, not diffuse through. Why don’t we see any accumulation in the membranes in Figure 9?

I can’t work out exactly the details of the comparison between the experimental data and the simulations so I will get in touch with the authors.

Hot (biochemistry-related) topics

I am in charge of a module entitled “Advanced Skills for Biochemistry“. Our third year Biochemistry (Honours) students take this course. One of their tasks is to prepare and present a poster on a hot topic or technique. I have therefore asked the world (via Twitter) and my colleagues at the Institute of Integrative Biology to come up with suggestions of topics for these posters, as well as references that students could use as a starting point.

[I have done so in previous years too].

  1. T cell quiescence and activation; suggested by Neill Liptrott. Reference: Metabolic coordination of T cell quiescence and activation; Chapman et al, 2019.
  2. Microbes and preeclampsia; suggested by Doug Kell. Reference: A Dormant Microbial Component in the Development of Preeclampsia; Kell & Kenny, 2016.
  3. How drugs get into cells; suggested by Doug Kell. How drugs get into cells: tested and testable predictions to help discriminate between transporter-mediated uptake and lipoidal bilayer diffusion; Kell & Oliver, 2014.
  4. Microbes and Alzheimer’s Disease; suggested by Doug Kell. Reference: Microbes and Alzheimer’s Disease; Itzhaki et al, 2016.
  5. Evolutionary covariance for protein structure prediction; suggested by Dan Rigden via email: “The topic of evolutionary covariance, with myriad uses but particularly for protein structure prediction, goes from strength to strength. Unfortunately, Google decided not to make the code available or (I think) to publish anything in a journal [there’s a bit of a separate lesson to the students there]. However, they can read about it here and here. This paper, out this week, is the most similar approach I’m aware of and works extremely well. It has a server (that the students could try…) and the code is available.”
  6. Synthetic biology for faster enzymes, suggested by Doug Kell. Reference: Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently. Currin et al, 2015.
  7. Nutraceuticals and longevity, suggested by Doug Kell. Reference: Prolonging healthy aging: Longevity vitamins and proteins; Ames, 2018.
  8. Mitochondrial Breakups, suggested by Violaine See. The Good and the Bad of Mitochondrial Breakups; Sprenger, 2019
  9. Signalling controlled by frequency modulation, suggested by Violaine Sée, e.g. this article.
  10. CryoEM – suggested by Steve Royle via Twitter; advances in electron detectors and software has led to explosion of new fascinating structures. Pat Eyers agrees and provides these examples of CryoEM of the anaphase promoting complex.
  11. Organoids cultures, suggested by Dada Pisconti, e.g. this review Modeling mouse and human development using organoid cultures
  12. Oxygen sensing across kingdoms, Masson et al; Conserved N-terminal cysteine dioxygenases transduce responses to hypoxia in animals and plants – see also 2019 Nobel Prize announcement.

Reply from Wolfgang Parak

Regular readers will remember a recent post where I documented the critical peer review of 20 influential (more than a 1000 citations) articles. I reviewed them initially in a Twitter thread and then also reproduced my comments on PubPeer.

Wolfgang Parak, corresponding author of one of these 20 papers (Cytotoxicity of colloidal CdSe and CdSe/ZnS nanoparticles), has kindly responded to my comments. I reproduce his response below (with his authorisation). The full discussion is here. Wolgang is doing the right thing. I hope other authors will follow suit; especially authors of papers where more serious concerns where raised such as here, here here, here, here, here, here and here.

Dear Raphael

thank you very much for your comments about our article.  In general I agree with your points. There were essentially two shortcomings you pointed out.

1) At low nanoparticle concentration there are less adherent cells after incubation than before incubation. In fact, at low nanoparticle concentrations there should be no effect, and thus the number of adherent cells before and after incubation should be equal (R = 1). However, cells proliferate. Thus, in case cells proliferate during incubation, there would be more cells after than before incubation (R > 1). In order to get rid of this effect we incubated the cells in SATO medium, which should stop cell proliferation. However, in fact the use of SATO medium also causes loss of some adherent cells. We pointed this out in our manuscript: “Exchanging the serum-containing cell culture medium to serum-free SATO medium resulted in the detachment of a significant fraction of cells, i.e., R < 1 even for c(Cd) = 0. Therefore, we assume that there are no toxic effects due to Cd in the first region of low Cd concentration and that the value of R < 1 can be simply explained by the effect of the SATO medium”. Thus, we did the control, that there is an effect due to changes of serum supplemented medium to the SATO medium which is supposed to stop cell proliferation. In retro-perspectives the conditions could have been better optimized, in getting culture conditions where there is no cell proliferation, but there is also no loss of cells during the incubation period. In addition, had I to plan the study today again, I would use a different assay which probes cell metabolism, instead of using cell adhesion as quantifier for toxicity. There are a variety of excellent assays, which also have different sensitivity, see for example: ” X. Ma, R. Hartmann, D. Jimenez de Aberasturi, F. Yang, S. J. H. Soenen, B. B. Manshian, J. Franz, D. Valdeperez, B. Pelaz, N. Feliu, N. Hampp, C. Riethmüller, H. Vieker, N. Freese, A. Gölzhäuser, M. Simonich, R. Tanguay, X.-J. Liang, W. J. Parak, “Colloidal Gold Nanoparticles Induce Changes in Cellular and Subcellular Morphology”, ACS Nano 11, 7807−7820 (2017)”. The reason why at that time we used the adhesion assay was that we were afraid that some metal ions may interfere with colorimetric viability assays. In a previous work (C. Kirchner, M. George, B. Stein, W. J. Parak, H. E. Gaub, M. Seitz, “Corrosion protection and long-term chemical functionalization of gallium arsenide in aqueous environment”, Advanced Functional Materials 12, 266-276 (2002)) we had observed that the MTT assay interferes with As ions released from GaAs surfaces and thus at that time we decided against the use of a biochemical assay.

2) The patch clamp experiment, in which ion currents were measured with and without presence of nanoparticles was merely quantitative. We thought at this time that it would be a good add-on to the paper, using one more complementary technique apart from the detachment assay to probe for toxic effects. In literature at that time there were reports suggesting the use of quantum dots for cell labelling. In our detachment assay we had shown, that at high concentration quantum dots are toxic to cells. Our motivation was to show, that one can use low quantum dot concentrations, which are enough to label cells, but which are low enough not to cause acute toxicity. Thus, we chose one concentration of quantum dots, which is enough to label cells, and showed that with this concentration ion channel currents were not affected. In retro-perspectives with some additional work we could have made also a more quantitative investigation, with a variation of quantum dot concentration, to see at which concentration ion channels are affected.

I hope with this I could comment on your two major points.

Best wishes


(Wolfgang Parak,

Nanomedicine on Planet F345

Last year, Matthew Faria et al published Minimum information reporting in bio–nano experimental literature, introducing a checklist (MIRIBEL) of experimental characterisations that should accompany any new research paper. 12 months later, the same journal has published 22 (!!!) short opinion pieces. As I feel particularly generous (and a bit facetious) today, I shall summarise those 22 pieces in 2 sentences.

  1. There are authors who feel that MIRIBEL is great and should be implemented although really colleagues should also consider using these other characterization techniques (that they happen to be developing/proposing in their lab/European network [INSERT ACRONYM]).
  2. There are authors who think that there is a risk that MIRIBEL standardisation will stiffle creativity and innovation  (and they also regret that MIRIBEL authors haven’t cited their editorials deploring irreproducible research).

Thankfully, there are more interesting takes from young researchers on Twitter (why do we need journals again?).

Wilson Poon remarks that the sheer amount of acronyms for nano-bio related guidelines & databases is insane;  he remains unconvinced that making new guidelines is the best way to address the current “significant barriers to progress in [nanomedicine],  and, even more damningly, he notes the hypocrisy of many researchers in the field [who] just talk the talk, and not walk the walk

Shrey Sindhwani demands quantification of what is happening to particles at a cellular and sub-cellular level, multiple lines of evidence and the use of appropriate biological controls. He makes two other really important points: 1) he demands critical discussion of what is in the literature; 2) he says we need replications: multiple groups should try to reproduce core concepts of the field for their systems. This involves mechanistic studies of what the body does to your specific formulation. This will define the scope of a broad concept and its applicability.

I largely agree with Wilson and Shrey. MIRIBEL may be well intentioned (and so are most responses), but they are not digging in the right place, and that is because they might otherwise find skeletons that they’d rather not find. This is very explicit in the original MIRIBEL paper:

… our intention is not to criticize existing work or suggest a specific direction for future research. The absence of standards and consistency in experimental reporting is a systemic problem across the field, and our own work is no exception.

God forbids criticizing existing work. If we start there, people might even consider criticising our own work and then where we will it stop? We might have to answer difficult questions at conferences?! That would be scientific terrorism.

Better reporting guidelines is not the solution because it does not address the core of the problems we are facing. In his 2012 paper entitled “Why Science Is Not Necessarily Self-correcting, John P. A. Ioannidis noted that

Checklists for reporting may promote spurious behaviors from authors who may write up spurious methods and design details simply to satisfy the requirements of having done a good study that is reported in full detail; flaws in the design and execution of the study may be buried under such normative responses.

This is exactly what will happen with MIRIBEL. Some will ignore it. Some will talk the talk, i.e. they will burry flaws in the design and execution of the study under a fully checked list of characterizations. Ben Ouyang makes a similar point when he asks what’s the point of reporting standards that might not relate to the problem?:

So, what are the core issues. What needs to be done?

First, we need to look critically at the scientific record. We need to sort out our field. We need to know what are solid concepts we can build on and what are fantasies that have been pushed at some point to get funding but have no underpinnings in the real world. This is important and necessary work. It may impact evaluation of what is worth or not worth funding. It may impact evaluation of risks and public perception of science and technology (badly needed) and even the approval of clinical trials. It may make all the difference for a starting PhD student if she finds a critical analysis of the paper their supervisor is asking them to base their PhD project on.

I have started here with 20 reviews of highly cited papers; we need more people joining in this effort of critically annotating the literature. The tools are available via PubPeer (have you installed their browser plugin that tells you when you are reading a paper which has comments available?). It is not accidental that such tools are not provided by the shiny journals such as Nature Nanotechnology who are happy to publish some buzz about reproducibility but have very little interest in correcting the scientific record.

We need clarity and critical thinking. We need to evaluate what we have. Take one of the founding idea of bionano, that nanoparticles are good at crossing biological barriers. Where does this idea comes from? What does it actually mean (i.e. what % of particles do that? which barriers are we talking about? “Good” compared to what?)? What is the evidence? Is it true? Can it be tested? Are we being good scientists when we make such statements in the introduction of our papers, in press releases or in grant applications? I would argue, contrarily to Ben, that the problem is not that things are complex, but rather that we have been blurring simple facts under a ton of mud for about two decades [1].

In his 2012 paper already cited above, Ioannidis describes science on planet Planet F345, Andromeda Galaxy, Year 3045268. It sounds worryingly not exotic. Let’s try not to emulate Planet F345.

Planet F345 in the Andromeda galaxy is inhabited by a highly intelligent humanoid species very similar to Homo sapiens sapiens. Here is the situation of science in the year 3045268 in that planet. Although there is considerable growth and diversity of scientific fields, the lion’s share of the research enterprise is conducted in a relatively limited number of very popular fields, each one of that attracting the efforts of tens of thousands of investigators and including hundreds of thousands of papers. Based on what we know from other civilizations in other galaxies, the majority of these fields are null fields—that is, fields where empirically it has been shown that there are very few or even no genuine nonnull effects to be discovered, thus whatever claims for discovery are made are mostly just the result of random error, bias, or both. The produced discoveries are just estimating the net bias operating in each of these null fields. Examples of such null fields are nutribogus epidemiology, pompompomics, social psychojunkology, and all the multifarious disciplines of brown cockroach research—brown cockroaches are considered to provide adequate models that can be readily extended to humanoids. Unfortunately, F345 scientists do not know that these are null fields and don’t even suspect that they are wasting their effort and their lives in these scientific bubbles.

Young investigators are taught early on that the only thing that matters is making new discoveries and finding statistically significant results at all cost. In a typical research team at any prestigious university in F345, dozens of pre-docs and post-docs sit day and night in front of their powerful computers in a common hall perpetually data dredging through huge databases. Whoever gets an extraordinary enough omega value (a number derived from some sort of statistical selection process) runs to the office of the senior investigator and proposes to write and submit a manuscript. The senior investigator gets all these glaring results and then allows only the manuscripts with the most extravagant results to move forward. The most prestigious journals do the same. Funding agencies do the same. Universities are practically run by financial officers that know nothing about science (and couldn’t care less about it), but are strong at maximizing financial gains. University presidents, provosts, and deans are mostly puppets good enough only for commencement speeches and other boring ceremonies and for making enthusiastic statements about new discoveries of that sort made at their institutions. Most of the financial officers of research institutions are recruited after successful careers as real estate agents, managers in supermarket chains, or employees in other corporate structures where they have proven that they can cut cost and make more money for their companies. Researchers advance if they make more extreme, extravagant claims and thus publish extravagant results, which get more funding even though almost all of them are wrong.

No one is interested in replicating anything in F345. Replication is considered a despicable exercise suitable only for idiots capable only of me-too mimicking, and it is definitely not serious science. The members of the royal and national academies of science are those who are most successful and prolific in the process of producing wrong results. Several types of research are conducted by industry, and in some fields such as clinical medicine this is almost always the case. The main motive is again to get extravagant results, so as to license new medical treatments, tests, and other technology and make more money, even though these treatments don’t really work. Studies are designed in a way so as to make sure that they will produce results with good enough omega values or at least allow some manipulation to produce nice-looking omega values.

Simple citizens are bombarded from the mass media on a daily basis with announcements about new discoveries, although no serious discovery has been made in F345 for many years now. Critical thinking and questioning is generally discredited in most countries in F345.

[1] The example of uptake of nanoparticles in cells is a case in point. Endocytosis was literally discovered and initially characterized using gold colloids as electron microscopy contrast agents in the 1950s and 1960s, yet half a century later, tens of thousands of articles write that the uptake of nanoparticles in cells is a mystery that urgently needs to be investigated.

20 critical reviews of influential articles about nanoparticles and cells

I have commented on the 20 highly cited articles below. They all relate to nanoparticles and cells. They were published between 1998 and 2006 and have received more than 1,000 citations each, over 40,000 citations overall.

I have used Twitter to document my reviewing process.

I have copied all of my reviews to PubPeer ; see the link below each papers in the bibliography at the bottom of this post. The orange colour indicates serious problems; the blue colour indicates that important old relevant papers have been overlooked.

You can also find the tweets via the ThreadReaderApp:


1             Bruchez, M., Moronne, M., Gin, P., Weiss, S. & Alivisatos, A. P. Semiconductor nanocrystals as fluorescent biological labels. Science 281, 2013-2016, doi:10.1126/science.281.5385.2013 (1998).

=> Comment on PubPeer.

2             Gref, R. et al. ‘Stealth’ corona-core nanoparticles surface modified by polyethylene glycol (PEG): influences of the corona (PEG chain length and surface density) and of the core composition on phagocytic uptake and plasma protein adsorption. Colloids and Surfaces B-Biointerfaces 18, 301-313, doi:10.1016/s0927-7765(99)00156-3 (2000).

=> Comment on PubPeer.

3             Lewin, M. et al. Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nature Biotechnology 18, 410-414, doi:10.1038/74464 (2000).

=> Comment on PubPeer.

4             Akerman, M. E., Chan, W. C. W., Laakkonen, P., Bhatia, S. N. & Ruoslahti, E. Nanocrystal targeting in vivo. Proceedings of the National Academy of Sciences of the United States of America 99, 12617-12621, doi:10.1073/pnas.152463399 (2002).

=> Comment on PubPeer.

5             Hirsch, L. R. et al. Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance. Proceedings of the National Academy of Sciences of the United States of America 100, 13549-13554, doi:10.1073/pnas.2232479100 (2003).

=> Comment on PubPeer.

6             Lai, C. Y. et al. A mesoporous silica nanosphere-based carrier system with chemically removable CdS nanoparticle caps for stimuli-responsive controlled release of neurotransmitters and drug molecules. Journal of the American Chemical Society 125, 4451-4459, doi:10.1021/ja028650l (2003).

=> Comment on PubPeer.

7             Wu, X. Y. et al. Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nature Biotechnology 21, 41-46, doi:10.1038/nbt764 (2003).

=> Comment on PubPeer.

8             Gao, X. H., Cui, Y. Y., Levenson, R. M., Chung, L. W. K. & Nie, S. M. In vivo cancer targeting and imaging with semiconductor quantum dots. Nature Biotechnology 22, 969-976, doi:10.1038/nbt994 (2004).

=> Comment on PubPeer.

9             Sondi, I. & Salopek-Sondi, B. Silver nanoparticles as antimicrobial agent: a case study on E-coli as a model for Gram-negative bacteria. Journal of Colloid and Interface Science 275, 177-182, doi:10.1016/j.jcis.2004.02.012 (2004).

=> Comment on PubPeer.

10           Connor, E. E., Mwamuka, J., Gole, A., Murphy, C. J. & Wyatt, M. D. Gold nanoparticles are taken up by human cells but do not cause acute cytotoxicity. Small 1, 325-327, doi:10.1002/smll.200400093 (2005).

=> Comment on PubPeer.

11           El-Sayed, I. H., Huang, X. H. & El-Sayed, M. A. Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: Applications in oral cancer. Nano Letters 5, 829-834, doi:10.1021/nl050074e (2005).

=> Comment on PubPeer.

12           Hussain, S. M., Hess, K. L., Gearhart, J. M., Geiss, K. T. & Schlager, J. J. In vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicology in Vitro 19, 975-983, doi:10.1016/j.tiv.2005.06.034 (2005).

=> Comment on Pubpeer.

13           Kirchner, C. et al. Cytotoxicity of colloidal CdSe and CdSe/ZnS nanoparticles. Nano Letters 5, 331-338, doi:10.1021/nl047996m (2005).

=> Comment on PubPeer.

14           Loo, C., Lowery, A., Halas, N. J., West, J. & Drezek, R. Immunotargeted nanoshells for integrated cancer imaging and therapy. Nano Letters 5, 709-711, doi:10.1021/nl050127s (2005).

=> Comment on PubPeer.

15           Morones, J. R. et al. The bactericidal effect of silver nanoparticles. Nanotechnology 16, 2346-2353, doi:10.1088/0957-4484/16/10/059 (2005).

=> Comment on PubPeer.

16           Chithrani, B. D., Ghazani, A. A. & Chan, W. C. W. Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Letters 6, 662-668, doi:10.1021/nl052396o (2006).

=> Comment on PubPeer.

17           Huang, X. H., El-Sayed, I. H., Qian, W. & El-Sayed, M. A. Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods. Journal of the American Chemical Society 128, 2115-2120, doi:10.1021/ja057254a (2006).

=> Comment on PubPeer.

18           Panacek, A. et al. Silver colloid nanoparticles: Synthesis, characterization, and their antibacterial activity. Journal of Physical Chemistry B 110, 16248-16253, doi:10.1021/jp063826h (2006).

=> Comment on PubPeer.

19           Rosi, N. L. et al. Oligonucleotide-modified gold nanoparticles for intracellular gene regulation. Science 312, 1027-1030, doi:10.1126/science.1125559 (2006).

=> Comment on PubPeer.

20           Xia, T. et al. Comparison of the abilities of ambient and manufactured nanoparticles to induce cellular toxicity according to an oxidative stress paradigm. Nano Letters 6, 1794-1807, doi:10.1021/nl061025k (2006).

=> Comment on PubPeer.