82% self-citation and grave misrepresentation in an ACS Nano Focus article

This post is cross-posted at PubPeer.

Mirkin and Petrosko review with enthusiasm and even a certain amount of lyricism the properties and applications of Spherical Nucleic Acids, an expression coined by Mirkin to describe particles prepared in the Mirkin group. Out of the 196 cited references, 160 or 82% have Mirkin as an author. Of the 36 other references, 16 are reviews, books or pre-1920 articles. Of the 20 remaining references, 9 are by Mirkin’s former PhD students or former post-doctoral researchers, and one is the clinical trial of Mirkin’s nanoparticles led by a Northwestern colleague. Thus out of 196 references, 10 or 5% are dedicated to non-Mirkin related original contributions to the topic covered in the Nano Focus (Figure 1). One might wonder whether the Editor-in-Chief (another Mirkin group alumnus) and the editorial board have a view on when an excessive amount of self-citations becomes problematic.

Figure 1: Classifications of the references in the Nano Focus article by Mirkin and Petrosko

My main concern is however not the excessive (?) self-citation but the grave misrepresentation. Table 1 documents the discrepancy between the text in the paragraph “Intracellular Diagnostics and Therapeutics” and what happened in reality. The success story narrated by Mirkin and Petrosko hides a commercialisation disaster (the SmartFlares) and repeated clinical applications failures resulting in hundreds of millions of dollars of public and private investments being wasted as well as false hopes given to patients. Those investments and hopes have been fuelled by scientific articles, which, like this one, hype results and ignore or downplay inconvenient facts, and in particular the fundamental limitation that endosomal escape represents for intracellular applications of nanoparticles. The story even includes research fraud in the Mirkin-founded company Exicure.

Table 1: Misrepresentations in the paragraph “Intracellular Diagnostics and Therapeutics” of Mirkin and Petrosko Nano Focus

Text reproduced from Mirkin and Petrosko	Comment
In the biomedical space, in 2006, we made the remarkable discovery that, when SNA-gold nanoparticle conjugates were introduced into cell culture (epithelial cells), the cells actively internalized them. (75) Through careful inhibition- and knockdown-studies, it was determined that the SNAs were taken up by scavenger receptor (class A)- and caveolin-mediated endocytosis in high quantities unlike their nanoparticle-free DNA counterparts. (76,77) Later, it was shown that this characteristic is general for SNAs, spanning over 60 different cell types, including stem cells. These nanostructures also resisted enzymatic degradation (78) and did not elicit an adverse immune response. (79) Significantly, we now had a way to use SNAs to measure and manipulate intracellular, not just extracellular, contents. This discovery led to the swift development of DNA-functionalized nanomaterials as gene regulation agents (70,75) and intracellular probes. (80)	The paragraph seems to suggest that first they 1) discovered the supposedly special property of active internalisation, 2) then did lots of careful studies, 3) which finally gave them a way to manipulate intracellular concents.   But the big claim in the first article, Ref 75 (Rosi et al, Science, 2006) is notactive internalisation, but, as its title indicates, Intracellular Gene Regulation. Thus, contrarily to what this paragraph seems to suggest, Mirkin claimed intracellular regulation already in the first article and not after “careful inhibition- and knockdown-studies” (Ref 76-79 are between 2009 and 2013). This is important because the most remarkable feature of Rosi et al, the foundational 2006 article that claims intracellular regulation, is that it lacks any discussion of the mechanism of uptake of the particles or of the crucial issue of endosomal localisation and endosomal escape.
Specifically, DNA- and RNA-functionalized nanoparticles were found to be potent agents for gene regulation in antisense and RNAi pathways, respectively. These particles were designed to downregulate the expression of proteins associated with cancer in cells, tissues, animals, and ultimately humans. Because these particles were found to actively cross dermal, blood–brain, and blood–tumor barriers, skin (81) and brain cancers (82) were models for initial demonstrations. Teaming up with Amy Paller and Alex Stegh, we explored their potential in these arenas and later in the context of a variety of other cancers as well. The first-in-human clinical trials of nucleic acid-functionalized nanoparticles of this type, which targeted glioblastoma (83) and inflammatory markers in the skin associated with psoriasis, were run by Northwestern University and start-up companies spun out of it.	This paragraph severely misrepresents the status of SNAs clinical trials by omitting critical information, namely the results and eventual follow-up of those clinical trials, and the fate of the Mirkin-founded company Exicure. NU-0129 for the treatment of glioblastoma; the Northwestern-led phase 0 clinical trial mentioned in the paragraph (ref 83) included 8 patients. It ended five years ago in September 2018. I could not find evidence of any follow-up.AST-005 for the treatment of Mild to Moderate Psoriasis; Exicure; phase 1b completed in 2018 as part of a collaboration with Purdue Pharma. The study did not result in any statistically significant indications of efficacy and Purdue Pharma notified Exicure it has declined to exercise its option to develop AST-005 at this time (https://www.sec.gov/Archives/edgar/data/1698530/000169853018000016/a8-k43018exhibit991.htm) AST-008; Exicure; phase 1 (completed) and phase 1b/2 started and then was discontinued for “administrative reasons” ; 57 participants were enrolled, at least 26 patients dosed (https://classic.clinicaltrials.gov/ct2/show/NCT03684785); Press release Dec 10, 2021: Discontinuation of further enrollment and the ethical wind down of the Company’s ongoing Phase 1b/2 cavrotolimod (AST-008) clinical trial in patients with solid tumorsXCUR-FXN for the treatment of neurological disorder Friedreich’s Ataxia; Exicure; A clinical trial had been announced for 2022 but the same Dec 2021 press release tells us that there is an indefinite suspension of further development of the company’s XCUR-FXN program.Exicure, the company founded by Mirkin and others to develop the biomedical applications of SNAs, managed to attract hundreds of millions of dollars in grant funding, stock market financing and partnerships with pharma companies. Its value is currently close to zero after multiple failures to deliver combined with the revelation in November 2021 of a research fraud case within the company.
Moreover, DNA-functionalized particle-based intracellular detection of mRNA, (40,80,84,85) aptamers, (86) and other moieties in single, living cells was accomplished using “nanoflare” technology; a related system called the sticky-flare was developed for determining both the amount and spatial location of intracellular RNA. (87) Nanoflares were ultimately commercialized by AuraSense along with Merck/Millipore as Smart-Flares, and this platform was later enhanced through the development of FIT-flares. (88,89)	Again, this paragraph severely misrepresents the status of SNA-based intracellular detection through omitting critical facts, inconvenient articles, and also through mis-citations: A major shortcoming of the Mirkin studies cited is that they do not report how nanoparticles escape endosomes and which proportion of particles do so. There cannot be intracellular sensing without a large proportion of probes reaching their targets and intracellular RNAs are not in endosomes. The NanoFlares were indeed commercialised 10 years ago, but they are not available anymore since 2018, more than 5 years ago. In spite of an application potentially relevant to all cell biologist, global advertising and distribution, very few articles reported usage of the SmartFlares and the company eventually gave up. To the contrary, several articles (not cited by Mirkin and Petrosko) report that the SmartFlares are localised in endosomes and do not detect mRNAs, in particular our 2015 article The spherical nucleic acids mRNA detection paradox and Maria Czarnek & Joanna Bereta 2017 article SmartFlares fail to reflect their target transcripts levels. The Sticky-Flares were reported in a PNAS contributed paper (i.e. Mirkin chose the referees of his own article). Our letter to the Editor re-analysing the data and showing that the signal attributed to mRNA localisation instead comes from vesicular transport is available as a preprint: Sticky-flares: real-time tracking of mRNAs… or of endosomes? David Mason, and Raphaël Lévy (2015); https://doi.org/10.1101/029447 The Fit-Flares are presented as if they are an extension of the commercialised SmartFlares platform when in fact, 1) the SmartFlares are not commercialised anymore, and, 2) the Fit-Flares have nothing to do with SmartFlares. In fact, it is unclear why they are mentioned at all given that the Fit-Flares are not SNAs and not even nanoparticle-based!

Gold injections – how to use the scientific literature to sell snake oil to patients

To know more about the adventures of Dr Doxey, an unscrupulous charlatan ready to do anything to sell his worthless elixir, read the Lucky Luke Western album by Morris.

To know more about Goldic, a real story that does not happen in Lucky Luke’s imagined Wild West, but in the present time, in the UK, Germany and possibly other places, where doctors will take your money in exchange of a miraculous therapy where they will draw your blood and re-inject it after it has been incubated with gold nanoparticles, read Private Eye’s investigation featuring expert and former colleague Patricia Murray (and a quote from me too):

Khan made even wilder claims to a prospective patient: “It gets your body working as it did in your 20s. You will find yourself having more energy, you’ll be quicker in the way that you think – all those things are going to improve.

All of which is quackery, says Patricia Murray, professor of stem cell biology at Liverpool University. She told the Eye: “I am very concerned that this is being promoted as the next generation of stem cells. There is no evidence to support these claims. It seems patients are, once again, being exploited for financial gain.”

If you are in the UK, go and buy your copy in your local newsagent, or subscribe. For the others, I will, with the Eye’s generous authorisation, update this post and share the full article once the next issue of the Eye is out.

In the meantime, you can read a related PubPeer comment. Dr Doxey claimed the efficacy of his elixir based on “years of tireless research”. Present days charlatans mis-use the peer reviewed scientific literature to prove it.

Guest post: Rewarding Reproducibility and Correction in Science

This is a guest post by Jan-Philipp Günther, Max Planck Institute for Intelligente System.

During my PhD, I was involved in multiple scientific discussions, which focused on the reproducibility of scientific results. At first, we examined these results, which have been published in high-ranked journals, out of curiosity and to understand them further, but then discovered that the measurements were not reproducible. After determining the sources of errors (measurement artefacts), we published our results. This cost us a lot of time and energy and was mainly rewarded by positive feedback from colleagues at conferences, but I think that many scientists who try to understand and reproduce published results are not willing to make their findings public – and even if they try to do so, they might face resistance from publishers. I was also amazed to learn of other papers, which are known by several senior scientists of the community to be irreproducible, but where this insight never appeared in print. As scientists, we know that mistakes can and will happen and that science is merely the process of advancing our understanding. Hence publications that over time are no longer considered to be correct are a part of science, but to an even greater extent should repeating experiments and corrections of the literature be accepted and encouraged. These corrections can save time and money and in rare cases (e.g., medical sciences) even lives. In the current era of bibliometrics, the pressure on scientists to regularly produce high impact papers and the pressure on journals to publish the most spectacular results as fast as possible has led to an increasing occurrence of errors and in some cases even fraud (see this project as an example to address “Scientific Misconduct and the Attempt of a Counterattack”). Additionally, the current system rewards irreproducible publications with more citations, where in most cases the citing article does not even mention the replication failure. This makes me as a young scientist believe that action is needed to bring these problems to the awareness of scientists and publishers. During a recent scientific meeting with many fruitful discussions, I had the idea to encourage and reward scientists to revisit experiments with dedicated awards, especially if repeating experiments reveals new insights or is able to correct the literature. Some ideas for awards are listed below, which will hopefully start a discussion on this topic within the community.

Award for reproducing published experiments

An award could be dedicated to scientists, who spend time to reproduce challenging experiments. This might encourage scientists to test published results or make their findings public, if they did run the experiments already, but did not find the time to publish their results. These studies should be awarded independent of the outcome. The focus should be on the effort invested, the challenges and the impact of the results. The award should also consider the contribution of young scientists, which did the actual experiment, since this can in some cases be extremely time consuming and risky.

Advancing scientific reproducibility award

This award might be handed to individuals or organizations, which foster the progression of scientific reproducibility and corrections through continuous effort or single innovations. One example might be the establishment of an online tool for scientific exchange, a journal with generous correction policies, or a group of scientists, who fought a long time to correct a certain part of the literature against resistance. It is of course necessary to exclude ongoing scientific discussions, since it might be impossible to determine, which side is correct, or it might be impossible to find an impartial committee or reviewers for the award.

Self-correction award

Quite often the original authors of papers gain additional insights or have discovered a mistake, but the stigma of correcting their own work or retracting the original publication is too big. Although many scientists seem to feel this way, it is not what I found to be the case in the vast majority of discussions, which I had with colleagues, who are in favor of self-correction and retraction and do not regard this as a stigma. Until the opinion that self-correction is a noble (and necessary) act has not reached the majority of scientists, awards for such self-corrections could be implemented. The award should of course only be handed out, if the candidates are willing to accept it.

Award for anonymous whistleblowers

Awards for whistleblowers might already exist, but this award should be dedicated to anonymous whistleblowers, which lead to scientific corrections. Especially in the case of scientific misconduct, it might be impossible for one of the authors to correct erroneous publications oneself without the support of all coauthors. In this case the authors should be encouraged to alert other scientists anonymously with a public announcement. If this announcement leads to a correction of the scientific literature, the whistleblower can be nominated. The prize money should be donated to open science foundations, since it of course cannot be handed out to the awardee publically. Hence, the awardee is not benefiting, but rather the scientific community.

Best PubPeer comment

PubPeer.com has the potential to become a popular and valuable tool for scientific correction, but this might strongly depend on the culture of the scientific exchange. High quality and respectful comments could be encouraged with an award. This could also be implemented with a dedicated title, symbol or name tag on the website. Maybe only signed comments should be considered.

It would be beneficial if an independent institution could be founded to handle the awarding process. Application or nomination for the awards should be open to everyone. I hope this will lead to further discussion on the topic of scientific correction and will maybe someday help scientists to make the right decisions for the benefit of us all. The utopian dream of the smooth, perfect (self-)correction in science, may never be achieved, but we will hopefully be able to foster a culture, where reproducing experiments will be honored and where mistakes can be addressed in a respectful dialogue. Please let me know your comments and concerns, and please feel free to develop these ideas further.

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.

In this thread, I will be looking back at highly cited (over 1000 citations) ‘historical’ (prior to 2008) bionano papers, in particular those that deal with nanoparticles & cells. Comments most welcome. #bionano #NanoBubble

— (((Raphael Levy))) (@raphavisses) February 6, 2019

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:

• Intro and articles 1 to 10.
• Articles 11 to 17
• Articles 17 (continued) to 20

 

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.

 

 

A RESPONSE FROM CHAD MIRKIN’S GROUP [follow up #1/n]

Some readers might wonder why I am going on about this, so let me tell you: this is a considerably more important story than Stripy Nanoparticles Revisited. If, as I am arguing, some of this science is shaky, then it is not only the way we evaluate scientists and spend public money which are put into question, but the foundation of ongoing clinical trials.

Back to basics: in the section of Mirkin’s group PhD dissertation (previous post) that respond to our critique of their work on Spherical Nucleic Acid / SmartFlare / StickyFlare, they wrote the following:

Additionally, since the commercialization and sale of the nanoflare platform under the trade name Smartflare (Millipore), dozens of researchers around the world have participated in successful sequence-specific gene detection.[80]

Reference [80] correspond to six (half a dozen) articles, 80a to 80f (see below for details and links). Out of these six, only two are actual research papers, and, for both, the SmartFlares are a very minor addition to the work. Out of these two, only one is completely independent of Mirkin/EMD Millipore (the other one comes from Northwestern).

80a) is not primary research; it is an advertorial produced by EMD Millipore.

80b) is not primary research: it is a 300 words congress abstract (no figure). A follow up paper by the same group is discussed here.

80c) is a review and it is a collaboration between Northwestern (Mirkin’s University) and EMD Millipore. CoI statement from the paper: “D. Weldon is the R&D Manager at EMD Millipore responsible for the production of SmartFlares. Patents related to therapeutically targeting Nodal in tumor cells have been awarded to E.A. Seftor, R.E.B. Seftor, and M.J.C. Hendrix.”

80d) is a research paper. It does not show in any way that SmartFlares work. It assumes it does. The SmartFlare is a minor part of the article.

80e) is not primary research: it is an advertorial in a magazine funded by company advertising (including EMD Millipore in that very issue). The author is a journalist working for the magazine, not a practicing scientist.

80f) is a research paper. It does not show in any way that SmartFlares work. It assumes it does. SmartFlares are a very minor part of the article. The authors are from Northwestern, i.e. Mirkin’s University.

 

Is targeting your target?

Warren Chan’s group published in June a perspective in Nature Reviews Materials entitled “Analysis of nanoparticle delivery to tumours” (Wilhelm et al). A key finding of their analysis of the literature is the absence of increase in the (very small) amount of nanoparticles delivered to tumours in the past 10 years. In a welcome departure from the usually overly diplomatic and confused style that is the trademark of most scientific writing, Wilhelm et al write the following:

 “These advantages [of nanoparticles] have been dampened by the lack of translation to patient care, despite the large investment (more than $1 billion in North America in the past 10 years) and success in imaging and treating tumours in mouse models. As a result, nanomedicine has acquired a reputation of being “hype” that cannot deliver and has not transformed patient care as it promised 15 years ago”

[…]

“We must admit that our current approach is broken, and that is why we have not observed significant clinical translation of cancer nanomedicines. Many academic studies focused on the potential of nanoparticles for in vivo applications and showed that nanoparticles may be delivered to tumours by the EPR effect. However, publishing ‘proof of concept’ studies will only lead to curing mice and will unlikely translate to cancer care, irrespective of the number of nanoparticle design permutations used for cancer targeting studies.”

Recognising the magnitude of the challenge, Wilhelm et al propose a thirty year strategy for nanomedicine.

Not surprisingly the publication sparked a debate; see for example Derek Lowe’s blog “Nanoparticles Mix It Up With Reality” and the comments therein, and the article by Michael Torrice for Chemical and Engineering News “Does nanomedicine have a delivery problem?” which features a number of quotes by various nanomedicine players, some of whom contesting Wilhelm et al’s findings, or their relevance to the development of nanomedicine. The debate has also continued in the scientific literature with a comment by McNeil “Evaluation of nanomedicines: stick to the basics” and a response by Chan.

Another comment by Lammers et al has been published 10 days ago “Cancer Nanomedicine: Is targeting our target?”. The implicit answer of the authors is no, targeting is not our target and therefore the absence of progress noted by Wilhelm et al matters little. Lammers et al’s argument is first that the percentage of the injected dose reaching the tumour is not a good indicator of the potential of a therapy, and second, that nanomedicine has in fact had some successes even without targeting. To illustrate this latter point, their first example is Doxil, a liposomal formulation of the anti-cancer drug Doxurubicin.

It is rather unconvincing that Lammers et al would use Doxil as an indication of the success of nanomedicine given that it was developed in the 80s and 90s, i.e. one or two decades before the “nanomedicine” word had been coined and Clinton had announced the $500M National Nanotechnology Initiative (January 2000). A bibliography search for the word “nanomedicine” suggests that it started to be used in the year 2000, with this MIT Technology Review being one of the very first examples:

Nanomedicine Nears the Clinic

Minuscule “smart bombs” that find cancer cells, kill them with the help of lasers and report the kills. Sound crazy? Guess again. That treatment scenario may be less than a decade away.

by David Voss
January 1, 2000

Since this infamous MIT technology review, we have seen so many similar promises and so little translation that Chan’s review and the debate that it provoked are indeed an incredibly positive and much needed development.

There is another amusing thing about Lammers et al’s review. The title suggesting that targeting is not our target is further echoed in the conclusion as follows:

“Patients do not benefit from targeting as such, and a reported tumour accumulation of 0.7%ID does not mean that nanomedicines do not work. We have to think beyond targeting, and beyond numbers, and focus on carrier-dependent drugs, combination therapies, protocols for patient selection and ways to enable rapid and more efficient clinical translation.”

Yet targeting as such seems very much to have been the target of these authors as the (non-exhaustive) list of articles below illustrate.

1. Blume, G.; Cevc, G.; Crommelin, M.; Bakkerwoudenberg, I.; Kluft, C.;Storm, G.,Specific targeting with poly(ethylene glycol)-modified liposomes – coupling of homing devices to the ends of the polymeric chains combines effective target binding with long circulation times. Biochimica Et Biophysica Acta 1993, 1149 (1), 180-184.
2. Vingerhoeds, M. H.; Steerenberg, P. A.; Hendriks, J.; Dekker, L. C.; vanHoesel, Q.;Crommelin, D. J. A.; Storm, G., Immunoliposome-mediated targeting of doxorubicin to human ovarian carcinoma in vitro and in vivo. British Journal of Cancer 1996, 74 (7), 1023-1029.
   3. Storm, G.; Crommelin, D. J. A., Colloidal systems for tumor targeting. Hybridoma 1997, 16 (1), 119-125.
   4. Mastrobattista, E.; Koning, G. A.; Storm, G., Immunoliposomes for the targeted delivery of antitumor drugs. Advanced Drug Delivery Reviews 1999, 40 (1-2), 103-127.
   5. Mastrobattista, E.; Kapel, R. H. G.; Eggenhuisen, M. H.; Roholl, P. J. M.; Crommelin, D. J. A.; Hennink, W. E.; Storm, G., Lipid-coated polyplexes for targeted gene delivery to ovarian carcinoma cells. Cancer Gene Therapy 2001, 8 (6), 405-413.
   6. Mastrobattista, E.; Crommelin, D. J. A.; Wilschut, J.; Storm, G., Targeted liposomes for delivery of protein-based drugs into the cytoplasm of tumor cells. Journal of Liposome Research 2002, 12 (1-2), 57-65.
   7. Metselaar, J. M.; Bruin, P.; de Boer, L. W. T.; de Vringer, T.; Snel, C.; Oussoren, C.; Wauben, M. H. M.; Crommelin, D. J. A.; Storm, G.; Hennink, W. E., A novel family of L-amino acid-based biodegradable polymer-lipid conjugates for the development of long-circulating liposomes with effective drug-targeting capacity. Bioconjugate Chemistry 2003, 14 (6), 1156-1164.
   8. Metselaar, J. M.; Wauben, M. H. M.; Wagenaar-Hilbers, J. P. A.; Boerman, O. C.; Storm, G., Complete remission of experimental arthritis by joint targeting of glucocorticoids with long-circulating liposomes. Arthritis and Rheumatism 2003, 48 (7), 2059-2066.
   9. Schiffelers, R. M.; Koning, G. A.; ten Hagen, T. L. M.; Fens, M.; Schraa, A. J.; Janssen, A.; Kok, R. J.; Molema, G.; Storm, G., Anti-tumor efficacy of tumor vasculature-targeted liposomal doxorubicin. Journal of Controlled Release 2003, 91 (1-2), 115-122.
   10. Schmidt, J.; Metselaar, J. M.; Wauben, M. H. M.; Toyka, K. V.; Storm, G.; Gold, R., Drug targeting by long-circulating liposomal glucocorticosteroids increases therapeutic efficacy in a model of multiple sclerosis. Brain 2003, 126, 1895-1904.
   11. van Steenis, J. H.; van Maarseveen, E. M.; Verbaan, F. J.; Verrijk, R.; Crommelin, D. J. A.; Storm, G.; Hennink, W. E., Preparation and characterization of folate-targeted pEG-coated pDMAEMA-based polyplexes. Journal of Controlled Release 2003, 87 (1-3), 167-176.
   12. Mulder, W. J. M.; Strijkers, G. J.; Griffioen, A. W.; van Bloois, L.; Molema, G.; Storm, G.; Koning, G. A.; Nicolay, K., A liposomal system for contrast-enhanced magnetic resonance imaging of molecular targets. Bioconjugate Chemistry 2004, 15 (4), 799-806.
   13. Schiffelers, R. M.; Ansari, A.; Xu, J.; Zhou, Q.; Tang, Q. Q.; Storm, G.; Molema, G.; Lu, P. Y.; Scaria, P. V.; Woodle, M. C., Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Nucleic Acids Research 2004, 32 (19).
   14. Verbaan, F. J.; Oussoren, C.; Snel, C. J.; Crommelin, D. J. A.; Hennink, W. E.; Storm, G., Steric stabilization of poly(2-(dimethylamino)ethyt methacrytate)-based polyplexes mediates prolonged circulation and tumor targeting in mice. Journal of Gene Medicine 2004, 6 (1), 64-75.
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   16. Zhang, C. F.; Jugold, M.; Woenne, E. C.; Lammers, T.; Morgenstern, B.; Mueller, M. M.; Zentgraf, H.; Bock, M.; Eisenhut, M.; Semmler, W.; Kiessling, F., Specific targeting of tumor angiogenesis by RGD-conjugated ultrasmall superparamagnetic iron oxide particles using a clinical 1.5-T magnetic resonance scanner. Cancer Research 2007, 67 (4), 1555-1562.
   17. Dolman, M. E. M.; Fretz, M. M.; Segers, G. W.; Lacombe, M.; Prakash, J.; Storm, G.; Hennink, W. E.; Kok, R. J., Renal targeting of kinase inhibitors. International Journal of Pharmaceutics 2008, 364 (2), 249-257.
   18. Lammers, T.; Hennink, W. E.; Storm, G., Tumour-targeted nanomedicines: principles and practice. British Journal of Cancer 2008, 99 (3), 392-397.
   19. Lammers, T.; Subr, V.; Peschke, P.; Kuhnlein, P.; Hennink, W. E.; Ulbrich, K.; Kiessling, F.; Heilmann, M.; Debus, J.; Huber, P. E.; Storm, G., Image-guided and passively tumour-targeted polymeric nanomedicines for radiochemotherapy. British Journal of Cancer 2008, 99 (6), 900-910.
   20. Rijcken, C. J. F.; Schiffelers, R. M.; van Nostrum, C. F.; Hennink, W. E., Long circulating biodegradable polymeric micelles: Towards targeted drug delivery. Journal of Controlled Release 2008, 132 (3), E33-E35.
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   22. Mulder, W. J. M.; Castermans, K.; van Beijnum, J. R.; Egbrink, M.; Chin, P. T. K.; Fayad, Z. A.; Lowik, C.; Kaijzel, E. L.; Que, I.; Storm, G.; Strijkers, G. J.; Griffioen, A. W.; Nicolay, K., Molecular imaging of tumor angiogenesis using alpha v beta 3-integrin targeted multimodal quantum dots. Angiogenesis 2009, 12 (1), 17-24.
   23. Talelli, M.; Rijcken, C. J. F.; Lammers, T.; Seevinck, P. R.; Storm, G.; van Nostrum, C. F.; Hennink, W. E., Superparamagnetic Iron Oxide Nanoparticles Encapsulated in Biodegradable Thermosensitive Polymeric Micelles: Toward a Targeted Nanomedicine Suitable for Image-Guided Drug Delivery. Langmuir 2009, 25 (4), 2060-2067.
   24. Dolman, M. E. M.; Harmsen, S.; Storm, G.; Hennink, W. E.; Kok, R. J., Drug targeting to the kidney: Advances in the active targeting of therapeutics to proximal tubular cells. Advanced Drug Delivery Reviews 2010, 62 (14), 1344-1357.
   25. Lammers, T.; Subr, V.; Ulbrich, K.; Hennink, W. E.; Storm, G.; Kiessling, F., Polymeric nanomedicines for image-guided drug delivery and tumor-targeted combination therapy. Nano Today 2010, 5 (3), 197-212.
   26. Lammers, T.; Subr, V.; Ulbrich, K.; Peschke, P.; Huber, P. E.; Hennink, W. E.; Storm, G.; Kiessling, F., Long-Circulating and Passively Tumor-Targeted Polymer-Drug Conjugates Improve the Efficacy and Reduce the Toxicity of Radiochemotherapy. Advanced Engineering Materials 2010, 12 (9), B413-B421.
   27. Oerlemans, C.; Bult, W.; Bos, M.; Storm, G.; Nijsen, J. F. W.; Hennink, W. E., Polymeric Micelles in Anticancer Therapy: Targeting, Imaging and Triggered Release. Pharmaceutical Research 2010, 27 (12), 2569-2589.
   28. Talelli, M.; Iman, M.; Rijcken, C. J. F.; van Nostrum, C. F.; Hennink, W. E., Targeted core-crosslinked polymeric micelles with controlled release of covalently entrapped doxorubicin. Journal of Controlled Release 2010, 148 (1), E121-E122.
   29. van Rooy, I.; Cakir-Tascioglu, S.; Couraud, P. O.; Romero, I. A.; Weksler, B.; Storm, G.; Hennink, W. E.; Schiffelers, R. M.; Mastrobattista, E., Identification of Peptide Ligands for Targeting to the Blood-Brain Barrier. Pharmaceutical Research 2010, 27 (4), 673-682.
   30. Talelli, M.; Hennink, W. E., Thermosensitive polymeric micelles for targeted drug delivery. Nanomedicine 2011, 6 (7), 1245-1255.
   31. Talelli, M.; Rijcken, C. J. F.; Oliveira, S.; van der Meel, R.; Henegouwen, P.; Lammers, T.; van Nostrum, C. F.; Storm, G.; Hennink, W. E., Nanobody – Shell functionalized thermosensitive core-crosslinked polymeric micelles for active drug targeting. Journal of Controlled Release 2011, 151 (2), 183-192.
   32. van Rooy, I.; Mastrobattista, E.; Storm, G.; Hennink, W. E.; Schiffelers, R. M., Comparison of five different targeting ligands to enhance accumulation of liposomes into the brain. Journal of Controlled Release 2011, 150 (1), 30-36.
   33. Crielaard, B. J.; Lammers, T.; Schiffelers, R. M.; Storm, G., Drug targeting systems for inflammatory disease: One for all, all for one. Journal of Controlled Release 2012, 161 (2), 225-234.
   34. Crielaard, B. J.; Rijcken, C. J. F.; Quan, L. D.; van der Wal, S.; Altintas, I.; van der Pot, M.; Kruijtzer, J. A. W.; Liskamp, R. M. J.; Schiffelers, R. M.; van Nostrum, C. F.; Hennink, W. E.; Wang, D.; Lammers, T.; Storm, G., Glucocorticoid-Loaded Core-Cross-Linked Polymeric Micelles with Tailorable Release Kinetics for Targeted Therapy of Rheumatoid Arthritis. Angewandte Chemie-International Edition 2012, 51 (29), 7254-7258.
   35. Dolman, M. E. M.; Harmsen, S.; Pieters, E. H. E.; Sparidans, R. W.; Lacombe, M.; Szokol, B.; Orfi, L.; Keri, G.; Storm, G.; Hennink, W. E.; Kok, R. J., Targeting of a platinum-bound sunitinib analog to renal proximal tubular cells. International Journal of Nanomedicine 2012, 7, 417-433.
   36. Joshi, M. D.; Unger, W. J.; Storm, G.; van Kooyk, Y.; Mastrobattista, E., Targeting tumor antigens to dendritic cells using particulate carriers. Journal of Controlled Release 2012, 161 (1), 25-37.
   37. Kunjachan, S.; Jayapaul, J.; Mertens, M. E.; Storm, G.; Kiessling, F.; Lammers, T., Theranostic Systems and Strategies for Monitoring Nanomedicine-Mediated Drug Targeting. Current Pharmaceutical Biotechnology 2012, 13 (4), 609-622.
   38. Lammers, T.; Kiessling, F.; Hennink, W. E.; Storm, G., Drug targeting to tumors: Principles, pitfalls and (pre-) clinical progress. Journal of Controlled Release 2012, 161 (2), 175-187.
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   40. Talelli, M.; Oliveira, S.; Rijcken, C. J. F.; Pieters, E. H. E.; Etrych, T.; Ulbrich, K.; van Nostrum, R. C. F.; Storm, G.; Hennink, W. E.; Lammers, T., Intrinsically active nanobody-modified polymeric micelles for tumor-targeted combination therapy. Biomaterials 2013, 34 (4), 1255-1260.
   41. van der Meel, R.; Vehmeijer, L. J. C.; Kok, R. J.; Storm, G.; van Gaal, E. V. B., Ligand-targeted particulate nanomedicines undergoing clinical evaluation: Current status. Advanced Drug Delivery Reviews 2013, 65 (10), 1284-1298.
   42. Heukers, R.; Altintas, I.; Raghoenath, S.; De Zan, E.; Pepermans, R.; Roovers, R. C.; Haselberg, R.; Hennink, W. E.; Schiffelers, R. M.; Kok, R. J.; Henegouwen, P., Targeting hepatocyte growth factor receptor (Met) positive tumor cells using internalizing nanobody-decorated albumin nanoparticles. Biomaterials 2014, 35 (1), 601-610.
   43. Kunjachan, S.; Pola, R.; Gremse, F.; Theek, B.; Ehling, J.; Moeckel, D.; Hermanns-Sachweh, B.; Pechar, M.; Ulbrich, K.; Hennink, W. E.; Storm, G.; Lederle, W.; Kiessling, F.; Lammers, T., Passive versus Active Tumor Targeting Using RGD- and NGR-Modified Polymeric Nanomedicines. Nano Letters 2014, 14 (2), 972-981.
   44. Novo, L.; Mastrobattista, E.; van Nostrum, C. F.; Hennink, W. E., Targeted Decationized Polyplexes for Cell Specific Gene Delivery. Bioconjugate Chemistry 2014, 25 (4), 802-812.
   45. Theek, B.; Gremse, F.; Kunjachan, S.; Fokong, S.; Pola, R.; Pechar, M.; Deckers, R.; Storm, G.; Ehling, J.; Kiessling, F.; Lammers, T., Characterizing EPR-mediated passive drug targeting using contrast-enhanced functional ultrasound imaging. Journal of Controlled Release 2014, 182, 83-89.
   46. Liu, J.; Jiang, X. L.; Hennink, W. E.; Zhuo, R. X., A modular approach toward multifunctional supramolecular nanopolyplexes for targeting gene delivery. Journal of Controlled Release 2015, 213, E123-E124.
   47. Novo, L.; Takeda, K. M.; Petteta, T.; Dakwar, G. R.; van den Dikkenberg, J. B.; Remaut, K.; Braeckmans, K.; van Nostrum, C. F.; Mastrobattista, E.; Hennink, W. E., Targeted Decationized Polyplexes for siRNA Delivery. Molecular Pharmaceutics 2015, 12 (1), 150-161.
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A welcome Nature Editorial

I reproduce below a comment I have left on this Nature editorial entitled “Go forth and replicate!“.

Nature Publishing Group encouragement of replications and discussions of their own published studies is a very welcome move. Seven years ago, I wrote a letter (accompanying a submission) to the Editor of Nature Materials. The last paragraph of that letter read: “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.” [1]

The submission was “Stripy Nanoparticles Revisited” and it took three more years to publish it… in another journal; meanwhile Nature Materials continued to publish findings based on the original flawed paper [2]. The ensuing, finally public (after three years in the secret of peer review), discussions on blogs, news commentary and follow up articles were certainly very informative on the absolute necessity of changing the ways we do science to ensure a more rapid discussion of research results [3].

One of the lessons I draw from this adventure is that the traditional publishing system is, at best ill suited (e.g. Small: three years delay), or at worst (e.g. Nature Materials) completely reluctant at considering replications or challenges to their published findings. Therefore, I am now using PrePrints (e.g. to publish a letter PNAS won’t share with their readers [4]), PubPeer and journals such as ScienceOpen where publication happens immediately and peer review follows [5].

So whilst I warmly welcome this editorial, it will need a little more to convince me that it is not a complete waste of time to use the traditional channels to open discussions of published results.

[1] The rest of letter can be found at https://raphazlab.wordpress.com/2012/12/17/letter-to-naturematerials/
[2] The article was eventually published in Small (DOI:10.1002/smll.201001465

2 comments on PubPeer

); timeline: https://raphazlab.wordpress.com/2012/12/20/stripy-timeline/
[3] https://raphazlab.wordpress.com/stripy-outside/
[4] https://raphazlab.wordpress.com/2015/11/16/pnas-your-letter-does-not-contribute-significantly-to-the-discussion-of-this-paper/
[5] https://raphazlab.wordpress.com/2015/11/17/the-spherical-nucleic-acids-mrna-detection-paradox/

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.

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

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

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.