After months of efforts, my co-authors and I are absolutely delighted to share this preprint, which is special in many ways:
Said, Maha, Mustafa Gharib, Samia Zrig, and Raphaël Lévy. 2023. “Replication of “Carbon-dot-based Dual-emission Nanohybrid Produces a Ratiometric Fluorescent Sensor for in Vivo Imaging of Cellular Copper Ions”” OSF Preprints. November 29. doi:10.31219/osf.io/kf9qe.
This preprint is special because it does not contain any data*: it is a pre-registration of a study. This means that what you will read is not a selection of results assembled to tell a nice story, but our plans to test experimentally a series of hypothesis. We are submitting these plans for peer review, both formal (through PCI RR) and informal (everyone is invited to comment at PubPeer). This makes so much more sense than the traditional peer review system: by peer reviewing our proposed plans and methodology you can truly help us build a more robust study that will contribute to solve the paradox of intracellular sensing with nanoparticle probes and help establish standards in how to study endosomal escape of nanoparticles. Once the pre-registered report receives “In Principle Acceptance”, after one or more rounds of peer review, we will do the experimental work, following the registered protocol, and the results will be published whatever they are. So, not only does this approach helps achieve a sound methodology before the experiments starts, it also helps to solve the problem of publishing bias where “negative results” don’t get published thus distorting the literature.
This preprint is also special because it is the first public step in the ERC NanoBubbles replication project in which we hope to reproduce several highly cited articles that report intracellular sensing with nanoparticles. We will also use this mechanism of pre-registration of studies for the next replications.
Now, I am sure you are wondering how you can help? The good news is that there are many ways. Read our registered report. Share this post to give visibility to this initiative. Peer review the proposal and give us constructive feedback to improve our plans. Get in touch to help us with the next pre-registration where we want to do a multi-site replication and will therefore need partners (nanoparticle synthesis, characterisation, microscopy, image analysis).
I am incredibly grateful to Maha and Mustafa who have done most of the work preparing this document; to Samia who supervised Mustafa for a little bit of organic synthesis (the only bit that we have done pre-registration; see paper for details). We are also thankful to the European Research Council for funding the project, and to Nicole Hondow (University of Leeds) and Aurélien Deniaud (University of Grenoble) for their suggestions and comments on the manuscript.
Our work regarding the use of gold nanorods as contrast agents for photoacoustic tracking of stem cells has been just published (or here*). You can find all the technical details of the work there, so I will try to explain here the work for the readers who are not very familiar with our field.
It is important to have the appropriate tools to evaluate safety and efficacy of regenerative medicine therapies in preclinical models before they can be translated to the clinics. This is why there is an interest in developing new imaging technologies that enable real time cell tracking with improved sensitivity and/or resolution. This work is our contribution to this field.
To distinguish therapeutic cells from the patient’s own cells (or here from the mouse’s own cell), the therapeutic cells have to be labelled before they are implanted. It is well known, that biological tissue is more transparent to some regions of the light spectrum than others. This fact is very easy to try at home (or at your favourite club): if you put your hand under a green light, no light will go through it, whilst doing the same under a red light the result will be very different. That means that red light is less absorbed by our body. Near infrared light is even less absorbed and this is why this region of the spectrum is ideal for in vivo imaging. Therefore, we made our cells to absorb strongly in the near infrared so we can easily distinguish them.
Gold nanoparticles of different sizes and shapes (synthesis and picture by Joan Comenge).
To do this, we labelled cells with gold nanoparticles. Interestingly, the way gold nanoparticles interact with light depends on how their electrons oscillate. That means that size and shape of the nanoparticles determine their optical properties, and this is one of the reasons why we love to make different shapes of nanoparticles. In particular, gold nanorods strongly absorb in the near infrared and they are ideal contrast agents for in vivo imaging.
Figure reproduced from: The production of sound by radiant energy; Science 28 May 1881; DOI: 10.1126/science.os-2.49.242
We have now cells that interact with light in a different way than the tissue. The problem is that light is scattered by tissue, so resolution is rapidly lost as soon as you try to image depths beyond 1 mm. Obviously, this is not the best for in vivo imaging. Luckily for us, Alexander Graham Bell realised 130 years ago that matter emits sounds when is irradiated by a pulsed light. This is known as the photoacoustic effect and it has been exploited recently for bioimaging. Photoacoustic imaging combines the advantages of optical imaging (sensitivity, real-time acquisition, molecular imaging) and the good resolution of ultrasound imaging because ultrasounds (or phonons), contrarily to photons, are not scattered by biological tissue.
Silica-coated gold nanorods inside cells
To optimise the performance of our gold nanorods, we coated them with silica. Silica is glass and therefore it protects the gold core without interfering with its optical properties. This protection is required to maintain gold nanorods isolated inside cells since nanorods are entrapped in intracellular vesicles, where they are very packed. The absence of a protective coating ultimately would result in a broader and less intense absorbance band, which would be translated to a less intense photoacoustic signal and consequently a lower sensitivity in cell detection. This of special importance in our system, a photoacoustic imaging system developed by iThera Medical which uses a multiwavelength excitation to later deconvolute the spectral information of the image to find your components of interest. Thus, narrow absorption bands helps to improve the detection sensitivity even further. With this we demonstrated that we were able to monitor a few thousand nanorods labelled-cells with a very good 3D spatial resolution for 15 days. This allowed for example to see how a cell cluster changed with time, see how it grows or which regions of the cell cluster shows the highest cell density. In addition, this work opens the door to new opportunities such as multilabelling using gold nanorods of different sizes and consequently different optical properties to observe simultaneously different type of cells. We also believe that not only stem cell therapies, but also other fields that are interested in monitoring cells such as cancer biology or immunology can benefit from the advances described in our work.
You can find the original publication here (or here*).
All the datasets are available via Figshare.
That is the title of a paper now published at PloS One. I am particularly pleased at the publication of this paper and would like to congratulate and thank its authors. They have all moved on from Liverpool. The joint first authors are Umbreen Shaheen and Yann Cesbron (currently in Rennes, France). Third author is Paul Free, now at IMRE in Singapore. The data were previously published at Figshare (links below).
PS: As we are today the second of April and not the first, you can be confident that this is not an April Fool post.
This post is co-authored by Raphaël Lévy and Dave Mason.
Note: We contacted Chad Mirkin and EMD Millipore for comments. Chad Mirkin replied but did not allow me to share his comments as he prefers to discuss his work in peer reviewed manuscripts rather than blogs. EMD Millipore has provided a response (reproduced below) and is keen to further engage in the discussion. They wrote that they “look forward to responding to [the questions you pose at the end of the post] after your blog is posted so other researchers who may have the same questions can follow our discussion online.” [Update: they have gone into silent mode after that comment].
Update: The results of our study have now been published.
To image proteins in cells, biologists have powerful tools based on the Green Fluorescent Protein (GFP) for which Osamu Shimomura, Martin Chalfie and Roger Y. Tsien obtained the 2008 Nobel Prize in Chemistry. RNA molecules play crucial roles in cells such as coding, decoding, regulation, and expression of genes, yet they are much more difficult to study. SmartFlares are nanoparticle-based probes for the detection and imaging of RNA in live cells. Could they become the GFP of the RNA world?
Many certainly believe this to be the case. SmartFlare ranked second in TheScientist top ten 2013 innovations, with one of the judges, Kevin Lustig, commenting “These new RNA detection probes can be used to visualize RNA expression in live cells at the single-cell level.” The following year, SmartFlare won an R&D100 award. The technology comes from Chad Mirkin’s lab at Northwestern University. Chad Mirkin is the winner of numerous prestigious prizes and a science adviser to the President of the United States. The scientific articles introducing the SmartFlare concept (under the name of Nano-Flare) were published in the Journal of the American Chemical Society in 2007, ACS Nano in 2009, etc. In 2013, the SmartFlare technology was licensed to EMD Millipore. Here is one of their promotional video [Update 31/01/2022: this video is unfortunately not available anymore ; hopefully the text explanation below is still fully understandable]:
For a molecular sensor to work, it needs a detection mechanism. The principle of the SmartFlare is explained from 0:45. A capture oligonucleotide (i.e. DNA) is bound to the gold nanoparticles. A reporter strand is bound to the capture strand. The reporter strand carries a fluorophore but that fluorophore does not emit light because it is too close to the gold (the fluorescence is “quenched”). In the presence of the target RNA, the reporter strand is replaced by the target RNA and therefore released, quenching stops, and fluorescence is detected. The release is shown at 2:05. Simple and convincing. Gold nanoparticles are indeed excellent fluorescence quenchers (we have used this property in a couple of papers).
But, for a molecular sensor to work, it also needs to reach the molecule it is supposed to detect. The SmartFlares are shown at 1:40 entering the cells via endocytosis, a normal mechanism by which the cell engulfs extracellular material by entrapping them into a bag made of cell membrane. Molecules and particles which enter the cell by endocytosis normally remain trapped in this bag. This entrapping is essential to protect us from viruses and bacteria by preventing them from accessing the cell machinery. Here, however, at 1:45 – 1:46, something truly remarkable happens: the endosome (the bag) suddenly fades away leaving the particles free to diffuse in the cell and meet their RNA targets. This is a promotional video so you might say that the demonstration of, and explanation for, this remarkable endosomal escape is to be found in the primary literature but that is not the case.
There is an extensive body of literature (not related to SmartFlare) dealing with endosomal escape. Some bacteria (like Listeria which can cause food poisoning) and viruses (like Influenza or HIV) use proteins to destabilise the endosome, escape and cause disease. Other mechanisms involve altering the ion balance in the endosome to pop it like an over-inflated balloon (you can read more about the ‘Proton Sponge Effect’ in this review). The problem is that none of these scenarios are applicable to gold nanoparticles conjugated to oligonucleotides. The problem is compounded by the choice of techniques used to analyse SmartFlare uptake into cells. Most of the published papers (for examples see here, here and here) characterise “uptake” and do so largely via Flow Cytometry or Mass Spectrometry (to measure the gold content of the cells). These papers certainly support NanoFlares being taken up into endosomes, but don’t offer any evidence for endosomal escape. A systematic unbiased electron microscopy study would enable to gather an estimate of how many nanoparticles have escaped the endosomes. Alternatively, fluorescence microscopy can be used to visualise a diffuse (released) instead of punctate (still in endosomes) distribution of intensity. While there are some images of cells having taken up NanoFlares, the sort of resolution required to discern distribution is not afforded by publication-size figures.
Wouldn’t it be nice if we had access to the original data? Researchers are often left squinting at published figures and all too often have to rely on the author’s interpretation of the data. One solution to this problem is to make supporting data available after publication. This is the idea behind the JCB Dataviewer; allowing authors to upload the original data to support papers published in the Journal of Cell Biology. The other option is to make the data available before publication, in what is called Open Research. This has the huge advantage of opening up a discussion about data, its interpretation and meaning before going through the formal peer-review process.
It is this latter technique that we are currently using to share our study of the use of NanoFlares as VEGF RNA reporters in cells. Our Open Science Notebook gives an overview of the experimental design, results and discussion, while our OMERO server is being used to host all of the original data for anyone to access. The project is still in progress, however our main findings so far are that:
Treating the cells with DMOG, a compound known to increase VEGF RNA levels in the cells, doesn’t have any effect on the fluorescent intensity or distribution (scroll about half way down the results).
In all conditions where fluorescence is seen, the distribution is consistently punctate (see all of the data here ).
So far these findings have left us with several questions, the most interesting of which are:
Why do we see punctate fluorescence with the VEGF SmartFlares? If the SmartFlares are still in endosomes, they shouldn’t be able to interact with mRNA and thus fluorescence should be quenched.
Why do we see signal at all in the scrambled control?
Why do different cells take up varying amounts of SmartFlares? Fluid phase dextran shows approximately equal uptake in all cells.
We’re presently investigating these and other questions. As we find out more, we will continue to post the data and update the blog.
RESPONSE from EMD Millipore:
In their response, EMD Millipore pointed to a number of relevant publications suggesting that we should revise the post after having considered this evidence. We had already seen those references and we have not altered the post, but we reproduce EMD Millipore’s response below:
Oligo-modified nanoparticle internalization and endosomal release:
· This is thought to be the mechanism of release from endosomes (via osmotic pressure)
Observation of punctate fluorescence:
· At short time points, when these structures are indeed in the endosomes, or at low detection gains on a microscope (where you are adjusting for the brightest points) the staining appears punctate. (For example- the light in a room comes from the bulb, which is the brightest, but the room is still lit. Keeping only the brightest point in a picture would only show you the bulb.)
· Therefore, with regards to the experiment you’ve already performed, our first suggestion would be to turn up the gain to see cytoplasmic fluorescence.
It may be worth noting some of our more recent examples of SmartFlare in the literature, spanning across cancer and stem cell research on a variety of detection platforms (flow & microscopy). Here the punctate fluorescence is also observed, but you can also see nice cytoplasmic staining.
Collage of various images. Credits: top left “Shutterstock”, top right “Cathy Wilcox”, bottom left “Jeremy M. Lange for The New York Times, A scientist at Duke University measures silver nanoparticles”, bottom center “Terminator 3: Rise of the Machines – a vision surely now only decades away. Photograph: Observer”, bottom right “image courtesy of Oregon State University”. See links in the paragraph below for original publication.
I started my talk by asking the audience what these images had in common (I did point out that the one in the top right corner was, in my experience, scientifically accurate). The answer? These pictures had all been used to illustrate nanoparticle news in the previous week.
While these stories are very different, they all originate in peer reviewed scientific research.
The “inspired by Terminator” piece originally comes from a press release by the University of Michigan. The authors of the article and their PR department were seeking this kind of publicity: the original article (very interesting BTW) uses the word “bionic” and the press release starts with “Inspired by fictional cyborgs like Terminator…”. Good to get coverage but it is highly debatable whether this kind of analogies really help improve the general understanding of science.
Deborah Blum article is measured and well researched – as we would expect from thisaward-winning science journalist – and based on multiple interviews with scientists. Yet in some ways, it also reflects the deep problems we are facing with establishing solid evidence in support of scientific understanding, and, eventually, policy making.
Deborah Blum article quotes Elisabeth Loboa as saying that“There’s evidence that the particles penetrate into plasma membranes, and they can disrupt cell function” [link in the original article, which is excellent practice!]. The idea that nanoparticles can go through the membrane of cells is so often repeated that it must be true, right? Scientists making those sorts of claim should provide a very high level of proof (unfortunately, this does not happen during peer review) because there are at least two fundamental reasons to be highly skeptical of such claims, one related to evolution, and another one related to physical chemistry and thermodynamics.
Nanoparticles are of similar size to viruses. If viruses could so easily penetrate cells, we would not be here discussing the risk of nanoparticles. Thankfully, during evolution, cells have developed very advanced mechanisms to protect themselves from foreign objects. Viruses too have developed very advanced mechanisms to get in there. Quite simply none of the nanomaterials made in the lab today seriously approach the level of sophistication that viruses use to get access to the interior of the cell (see this movie for an example). The linked article by AshaRani et al provides no evidence of particles penetrating through the plasma membrane (apart from the table of content cartoon). The dose used in this particular study is huge: 200 micrograms of nanomaterials per mL of medium (the equivalent of 10 grams of silver for a 50 kg person). In line with many other studies (including our own work), AshaRani et al shownanoparticles overwhelmingly in endosomes, i.e. in bags inside cells where they are isolated from the cell machinery. Endosomal trapping also remains amajor limiting factor to siRNA delivery (even using nanoparticles).
Ignoring now the biology, at the simplest level, the membrane of cells is made of a bilayer of lipids. It has an hydrophobic interior and two hydrophilic surfaces. For an object to diffuse through the membrane, it would need to have no significant repulsive or attractive interactions with any of these components (otherwise it would be repelled and not go through, or attracted and then get stuck). It is hard to imagine any nanoparticle that would fulfill such criteria (see also post and comments here for more details). While it is unclear that any nanoparticle can diffuse through the membrane, many small molecules can. We therefore have this strange situation where the supposed capability of nanoparticles to go through the cell membrane is presented as a reason to be particularly worried even though this is unproven, unlikely for nanomaterials, and common for many smaller compounds (e.g. DAPI).
I am not blaming Deborah Blum nor Elisabeth Loboa for this confusion. Such statements have become the norm. Although a more detailed investigation would be necessary (and I am not qualified to do it though I’d be happy to collaborate), my hypothesis is that the confusion results from a combination of nano hype (both in terms of risks and potential applications – see the Terminator for one striking example), bias towards the publication of positive findings, absence of post-publication peer review and debate, and poor scientific standards in an interdisciplinary area where editors and referees often lack some of the skills to properly evaluate the work (e.g. material scientists with very little understanding of biology, etc).
The situation is however now extremely serious since it has reached the point where it affects understanding of science for both basic scientists and the general public. It is our responsibility to try to fix the system.
and, of course, our Editorial, which starts as follows,
Biological systems, albeit magnificently well organized on the nanometer scale, do not contain nanoparticles, least those of metals or semiconductors. This should be sufficient reason to question the need to dedicate a themed issue of Advanced Drug Delivery Reviews to “Biological Interactions of Nanoparticles”. What is new and needs to be highlighted?
That is the question… for the answer, you will have to read the Editoral and the articles!
Functional nanomaterials have recently attracted strong interest from the biology community, not only as potential drug delivery vehicles or diagnostic tools, but also as optical nanomaterials. This is illustrated by the explosion of publications in the field with more than 2,000 publications in the last 2 years (4,000 papers since 2000; from ISI Web of Knowledge, ‘nanoparticle and cell’ hit). Such a publication boom in this novel interdisciplinary field has resulted in papers of unequal standard, partly because it is challenging to assemble the required expertise in chemistry, physics, and biology in a single team. As an extreme example, several papers published in physical chemistry journals claim intracellular delivery of nanoparticles, but show pictures of cells that are, to the expert biologist, evidently dead (and therefore permeable). To attain proper cellular applications using nanomaterials, it is critical not only to achieve efficient delivery in healthy cells, but also to control the intracellular availability and the fate of the nanomaterial. This is still an open challenge that will only be met by innovative delivery methods combined with rigorous and quantitative characterization of the uptake and the fate of the nanoparticles. This review mainly focuses on gold nanoparticles and discusses the various approaches to nanoparticle delivery, including surface chemical modifications and several methods used to facilitate cellular uptake and endosomal escape. We will also review the main detection methods and how their optimum use can inform about intracellular localization, efficiency of delivery, and integrity of the surface capping.
A third highlight for our article on cellular uptake and fate of nanoparticle bioconjugates: after Nano Today and Nature Nanotechnology, Chemical Research in Toxicology editor Carol A Rouzer writes:
Nanoscience is generating considerable excitement by offering a diversity of new materials with a wide range of potential applications in chemistry, engineering, and biomedicine. However, the explosion of new nanoparticles raises concern regarding their environmental impact and potential toxicity. Many studies have investigated the toxicity of nanoparticles with emphasis on the influence of size, shape, and surface functionalization. However, it is important not only to describe which nanomaterials are toxic but also to understand their mechanisms of toxicity, a goal that requires a full understanding of how nanoparticles interact with cells. To that end Sée et al. [(2009) ACS Nano, 3, 2461] have explored the fate of peptide-coated gold nanoparticles in cells.
Today’s edition of ACS Nano includes our article on the fate of nanoparticles upon internalization in live cells. BBSRC has issued a Media release. Surely it won’t get this kind of coverage…
In the image below, the top section shows nanoparticles in a compartment called endosome in a cell (Transmission Electron Microscopy, fixed cells) while the bottom part is an overlay of bright field microscopy (grey) and photothermal microscopy (yellow-red). The photothermal microscopy allows the direct visualisation of the gold nanoparticles inside the cells. The cartoon on the right is a 3D representation of the enzyme cathepsin L (with a carefully chosen set of colours).
Rapha-z-lab 