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SmartFlare controversy: independent confirmation of endosomal localization

Check this previous post for a quick summary of the SmartFlare controversy, or read all SmartFlare-related posts if you are really passionate.

At the centre of the SmartFlare controversy is the rather simple question, from an experimental point of view, of how many Spherical Nucleic Acids (to use Chad Mirkin’s terminology), if any, escape the endosomal pathway.

In contradiction with Chad Mirkin’s many peer reviewed articles and EMD Millipore marketing material, we concluded (Mason et al, 2016) that the Spherical Nucleic Acids do not escape endosomes and do not detect cytosolic mRNAs.

A few days ago, Sven Budik et al, an Austrian group published their evaluation of the SmartFlare in the context of equine embryo development. They write: “In all positive cells,
regardless of whether they occurred in equine conceptus, trophoblastic vesicle or fibroblast cell culture, the fluorescence signal showed a spotted pattern that is in accordance with the observations of Mason et al. (2016).

They also used electron microscopy to look at the intracellular localization of the particles. Here is the relevant part of their discussion and conclusion (emphasis mine):

The present study indicates that the intracellular process of nanogold particle uptake is endocytic and endosomal with a lysosomal sorting after longer incubation periods. This finding is in agreement with results from HeLa cells in vitro (Gilleron et al. 2013). Similarly, nanoparticles injected intravenously were taken up by endocytosis and later
clustered in lysosomes primarily in macrophages (Sadauskas et al. 2007). The incorporation time of lipid nanoparticlecontaining short interfering RNA gold particles in HeLa cells was similar (Gilleron et al. 2013) to that demonstrated in equine trophoblast vesicles in the present study. Accumulation of SmartFlare probes in residual bodies may be a consequence of increased stability of the immobilised oligonucleotides adjacent to the nanogold particles due to enhanced nuclease resistance (Rosi et al. 2006). In accordance with the results of Mason et al. (2016), we observed no or very few nanogold particles free in the cytoplasm, confirming a primarily endosomal and lysosomal localisation.

This observation raises the question how a specific SmartFlare probe is able to detect its target mRNA located in the cytoplasm. One possible explanation for the generation of lysosome-located specific fluorescence signals by SmartFlare probes could be the existence of specific RNA sequences imported for subsequent degradation into lysosomes (Fujiwara et al. 2013). Further studies using qRT-PCR investigating the isolated lysosomal fraction before and after incubation with specific SmartFlare probes are necessary to confirm this hypothesis. An 18S RNA nano-flare probe had a dose-dependent cytotoxic effect on porcine fetal fibroblasts (Fu et al. 2016). In contrast, no cytotoxic effects or changes in morphology after incorporation of antisense oligonucleotide nanogold particles in a mouse endothelial cell line were observed by Rosi et al. (2006). In addition, in the present study, there was no evidence that incubation with the SmartFlare probes had a toxic effect on the equine cells tested, even at higher concentrations. This is in accordance with the results of Pan et al. (2009) demonstrating that 15-nm gold particles have only low cytotoxic effects compared with the detrimental effects of small 1.4-nm gold particles.

In conclusion, SmartFlare probes pass into early equine conceptuses at stages used for embryo transfer, as well as trophoblast vesicles and cells cultured in vitro. In these early ZP equine conceptuses, the time frame (.5 and ,24 h) for SmartFlare uptake would be suitable for practical applications in commercial embryo transfer programs. Therefore, these probes are suggested to be applicable to pre-implantation genetic diagnosis before transfer of these conceptuses to the recipient.

In summary, the authors’ results are entirely consistent with our observations. They conclude, quite reasonably, that if SmartFlares detect mRNAs whilst being in endosomes, they cannot directly detect cytosolic mRNAs. This is in direct contradiction with Mirkin et al and EMD Millipore. Then, they propose that if the SmartFlares work, they maybe detect mRNAs which are in endosomes. This an interesting hypothesis that will require further study and is very different from anything published by Mirkin and EMD Millipore (the relevant reference is here). Since Budik et al do not provide any evidence that the SmartFlares actually detect mRNAs in the first place, maybe a simpler explanation is that the SmartFlares signal is unspecific and result from the probe degradation by nucleases in endosomes.

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Thoughts on #LiveTweeting

Dave Mason on why you should be live-tweeting at conferences

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As a part of the Centre for Cell Imaging and a member of the Microscopy and BioImage Analysis community, I occasionally get away to conferences like the recent NEUBIAS training school and symposium in Portugal.

vessels_neubias_banner

Since having joined Twitter last year (@dn_mason), this is the second conference that I’ve been to, and as a result, was the second time I tried (with reasonable success) to Live Tweet at the conference.

Live What Now?

Going right back to basics, Twitter is a platform for broadcasting small messages (of ~140 characters). Some describe it as micro-blogging. To many, the brevity of each tweet is both it’s greatest strength and also one of the most frustrating features.

Live tweeting, is basically the act of providing a running commentary of a seminar, event or even a whole conference. All of the tweets associated with such an event can be tied together using…

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Publication bias. Grant bias.

All academics writing grants will tell you this: if you want to be successful when applying to a thematic research grant call, you must tick all of the boxes.

Now, imagine that you are a physicist, expert in quantum mechanics. A major funding opportunity arises, exactly matching your interest and track record. That is great news. Obviously you will apply. One difficulty however is that, amongst other things, the call specifies that your project should lead to the “development of highly sensitive approaches enabling the simultaneous determination of the exact position and momentum of a particle“.

At that point, you have three options. The first one is to write a super sexy proposal that somehow ignores the Heisenberg principle. The second option is to write a proposal that addresses the other priorities, but fudges around that particular specification, maybe even alluding to the Heisenberg principle. The third option is to renounce.

The first option is dishonest. The second option is more honest, but, in effect, is not so different from the third: your project is unlikely to get funded if you do not stick to the requirements of the call, as noted above. The third option demonstrates integrity but won’t help you with your career, nor, more importantly with doing any research at all.

And so, you have it. Thematic grant calls that ask for impossible achievements, nourished by publication bias and hype, further contribute to distortion of science.

OK, I’ll confess: I have had a major grant rejected. It was a beautiful EU project (whether BREXIT is partly to blame I do not know). It was not about quantum mechanics but about cell tracking. The call asked for simultaneous “detection of single cells and cell morphologies” and “non-invasive whole body monitoring (magnetic, optical) in large animals” which is just about as impossible as breaking the Heisenberg principle, albeit for less fundamental reasons. We went for option 2. We had a super strong team.

PhD student at the Frontiers in BioImaging conference

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65787098 Jennifer presents her poster at the conference

Guest post by Jennifer Francis, PhD student in the Institute of Integrative Biology.

I recently attended the Frontiers in BioImaging conference in London (14th-15th July 2016), organised by the Royal Microscopical Society (RMS). Since this highly specialised conference was relatively small, I got the opportunity to speak one-to-one with experts within the field of super-resolution microscopy about their cutting-edge imaging techniques. A number of microscopy companies, including Carl Zeiss and Leica, also showcased their latest products. The highlight of this trip, was presenting my poster entitled “Exploiting Fluctuations to Enhance Imaging Resolution of Biological Structures“, which generated lots of encouraging interest. Whilst in London, I also got the chance to explore the famous landmarks, whose architecture never fails to impress.

76544879As well as, attending the talks, I also sat in on the Annual General Meeting (AGM), where the…

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Chad Mirkin on Nano Hype

Chad Mirkin did a Reddit AMA yesterday (h/t Neil Withers).

Capture

 

(highlight mine)

Of these 1800 commercial products, 1700+ are in fact a single product, the famous Spherical Nucleic Acids/SmartFlares.

More on this blog and our paper (The spherical nucleic acids mRNA detection paradox) here.

With the risk of being accused of having cynical views…

502 citations and counting… but what do they mean? (if anything)

Evaluation of research and researchers is a perilous activity. Sometimes, the number of citations of a piece of work is used as an indicator. As my most cited article passed the 500 citations mark (according to Google Scholar), I have had a brief look at the 15 articles that, this year, cite our 2004 paper. Thanks to all colleagues who did read and, hopefully, enjoyed that paper.

The original article developed peptides as capping ligands for gold nanoparticles. The work included rational design of a peptide sequence (CALNN), observation of a small plasmon shift (2 nm) upon formation of the peptide self-assembled monolayer, analysis of systematic variations around that original sequence (some of which caused aggregation) and associated data analysis, including the use of an aggregation parameter.

Of the 15 articles that cited it in 2016:

  • One is a master thesis in Finnish.
  • Three were reviews (2, 10, 11).
  • Two seem to be pretty random citations (3, 7) and another one is relevant but not quite appropriate (5).
  • Two cited our work because they used a similar approach for their data analysis/interpretation (1, 14).
  • Four cited our work as an example of highly stable and biocompatible nanoparticle (4, 6, 8, 12).
  • and… ONE actually builds on our work by developing their own peptide sequence based on CALNN (13).

I am not quite completely sure what to conclude from this (limited) analysis. Comments welcome!

Articles are listed below with the context of the citation.

1. Priya A. Nair and K. Sreenivasan: “Non enzymatic colorimetric detection of glucose using cyanophenyl boronic acid included β-cyclodextrin stabilized gold nanoparticles”

The aggregation parameter was then determined from the equation (AA0)/A0, where A is the integrated absorbance between 550 and 650 nm for the system with glucose added and A0 is the integrated absorbance between 550 and 650 nm of the blank solution. A similar type of indicator has been used to analyze the aggregation of gold and silver particle induced aggregation in the presence of different analytes.23,24

2. J. Yang, Celina and B. Chithrani, Devika: “Nuclear Targeting of Gold Nanoparticles for Improved Therapeutics.”

(can’t access).

3. Adél Vágó et al: “One-step green synthesis of gold nanoparticles by mesophilic filamentous fungi” [incidentally, last two authors missing in the bibliography, sorry Mathias and Dave!]

Chemical and physical methods have certain drawbacks such as use of harsh chemicals, stringent synthesis conditions, energy and capital intensive production and formation of toxic residues [7], [8] and [9].

I suppose that could be construed as a criticism of our work (in which case it is welcome), or, alternatively, as a somewhat random citation to support what is an often made but pretty weak argument.

4. Yukiho HOSOMOMI et al: “Biocatalytic Formation of Gold Nanoparticles Decorated with Functional Proteins inside Recombinant Escherichia coli Cells”

Among these materals, protein-decorated Au NPs are of great importance because they can confer colloidal stability and bioactivity to Au NPs.[14, 15]

5. Hongyu Yang et al: “The reactivity study of peptide A3-capped gold and silver nanoparticles with heavy metal ions”

Among the series of metal binding peptides, peptide A3, with a sequence of AYYSGAPPMPPF, is one of the few sequences that can stabilize both gold and silver nanoparticles with a desirable control of the size, shape and composition [15]. It contains amino acids that are capable of interacting with both gold and silver surfaces by hydrogen bonding or hydrophobic interactions [16], [17] and [18].

[Our paper does not show this. Although our work is quite relevant to their study and could reasonably be cited, it is not appropriate in support of this statement…].

6. Jiaxue Gao et al: “Multiple detection of single nucleotide polymorphism by microarray-based resonance light scattering assay with enlarged gold nanoparticle probes”

During the last two decades, gold nanoparticles (GNPs) have been extensively employed for the development of ultrasensitive detection and imaging methods in analytical or biological sciences because GNPs have unique optical properties (i.e. surface plasma resonance (SPR) absorption and resonance light scattering (RLS)), a variety of surface coatings and great biocompatibility.33–40

7. Xiangqian Jia et al “Micromixer Based Preparation of Functionalized Liposomes and Targeting Drug Delivery”

As promising interface molecules, peptides may be a good choice of recognition element to modify liposomes in order to increase the targeting specificity.(22-26)

Pretty random citation?

8. Yong Li et al: “A biomimetic colorimetric logic gate system based on multi-functional peptide-mediated gold nanoparticle assembly”

Furthermore, through the rational design of peptide sequences, the peptide could recognize the target and mediate the dispersibility of AuNPs. Modulation of the dispersibility of AuNPs, accompanied by their plasmonic properties, changes the solution colour in an on–off/off–on manner.23–27

Very relevant and seems like a cool intriguing paper.

9. Abrin L. Schmucker et al. “Plasmonic paper: a porous and flexible substrate enabling nanoparticle-based combinatorial chemistry”

Consequently, developing surface modification strategies that also circumvent aggregation is necessary and researchers have gone to great lengths to address this challenge. Indeed, a current state-of-the-art “toolbox” of compatible reagents has been developed to impart stability and functionality which includes ligands such as DNA,24,25 polymers,26 peptides,27–29 etc.

10. Cláudia Couto et al. “Gold nanoparticles and bioconjugation: a pathway for proteomic applications”

The successful formation of SAMs can be monitored in the UV-Visible spectrum, as was the case of SAMs produced with a pentapeptide ligand that induced a shift of approximately 2 nm in the AuNPs plasmonic band.[40]

11. Annalisa Calò et al. “Nanoscale device architectures derived from biological assemblies: The case of tobacco mosaic virus and (apo)ferritin”

Inorganic building blocks of less than 10 nm size can be synthesized using two procedures, the capping method and the mold method. The capping method1–4) is mainly employed in nanoparticle (NP) synthesis and assembly and allows the production of structures, i.e., metal nanoparticles, surrounded by organic molecules,1–5) while the mold method allows the fabrication of building blocks of a tailored shape inside or outside a mold unit.6,7)

12. Akash Gupta et al. “Ultrastable and Biofunctionalizable Gold Nanoparticles”

One of the principal barriers to the industrial and general use of AuNPs is their poor capability to endure freeze-drying cycles.(23) This is one of the major reasons why most of the commercially available AuNPs are sold as solutions. In addition, many reports have shown that, when AuNPs are subjected to lyophilization,(42) larger AuNPs agglomerates are formed, and these aggregates cannot be dispersed again in solution.(43) Variations in the stabilizing agent help to control the shape and size of the final structures, though complete stability was achievable in only a few cases.(44, 45)

13. Yi Wang et al. “Smartphone spectrometer for colorimetric biosensing”

A specific peptide receptor (CALNN-Peg4-FYSHSFHENWPS)1,40 with high affinity to cTnl was synthesized and immobilized on the surface of 36 nm AuNPs through the cysteine residue (see the ESI† for the Experimental details). In the presence of cTnl, AuNPs aggregate due to bridging of peptides (on separate AuNPs) resulting in a red shift of the LSPR peak. The colour of the AuNP solution concomitantly changes from red to purple. After extensive aggregation the suspension becomes colourless because of precipitation of AuNP aggregates at the bottom of the cuvette. The average size of the aggregates formed, as revealed by dynamic light scattering (DLS), also increases with the time of incubation of cTnI (see Fig. S5 in the the ESI†).

Yes! That paper is actually one that not only cites our work but actually builds on our 2004 ms where we introduced the CALNN peptide sequence.

14. Pei Liu et al; “Gold nanoprobe functionalized with specific fusion protein selection from phage display and its application in rapid, selective and sensitive colorimetric biosensing of Staphylococcus aureus”

The spectra showed a slightly broadened plasmon band, which was red-shifted from 523 to 525 nm after protein modification (Fig. 2C). The red-shift is induced by the introduction of the protein layer (Levy et al., 2004).