Lighting up biological tissue with rotaxanes

2 05 2012

Water-soluble, deep-red fluorescent squaraine rotaxanes

Erin L. Cole ,  Easwaran Arunkumar ,  Shuzhang Xiao ,  Bryan A. Smith & Bradley D. Smith

Org. Biomol. Chem., 2012, Advance Article; DOI: 10.1039/c2ob06783h

By Loruhama M. Delgado

Squaraines are fluorescent dye molecules that emit in the red or near infrared region. Although, these dyes have been extensively studied, their use for biological imaging applications are limited by their susceptibility to nucleophilic attack and their propensity for hydrolysis in water. For this reason Smith and coworkers encapsulated squaraine dyes in macrocyclic tetralactams to produce squaraine rotaxanes. The encapsulation stabilizes the squaraines and prevents fluorescence quenching by self-aggregation.

They synthesized several rotaxanes by using a Leigh-type clipping reaction to trap the squaraines inside macrocyclic tetralactams. After that, they used click chemistry to attach protected polar groups to the ends of the squaraine, followed by a deprotection to produce eight different water-soluble rotaxanes.

They measured the stability of their compounds by monitoring their absorbance in water, and in 10% fetal bovine serum (FBS).  These studies showed that rotaxanes containing two or three stoppers groups attached to the ends of the squaraines were less stable than those containing four groups. Out of all the rotaxanes, only two that contained four stoppers groups were kept for future screening. These two compounds showed great stability in a pH range of 6-10. They also observed that these two compounds didn’t show any affinity to the protein albumin, suggesting that these compounds could be used as non-targeted tracers.

For this purpose, they performed in vivo studies with mice to compare two squaraine rotaxanes with Indocyanine Green (ICG), which is another non-targeted tracer. They took optical images of mice organs, which showed that the two rotaxanes signals were mostly in the bladder, while the one from ICG was located in the intestines. They then proceeded to compare the two rotaxanes by ex vivo fluorescence using excised organs. In these studies they found out that one of the rotaxanes tracer showed more pixel intensity values than the other one, but that both of these compounds showed very low retention in the tissues of the organs in mice.

I think that overall this communication was interesting. The narrative was engaging, the authors did a good job explaining their findings, and the paper was of appropriate length. Although they were able to get water-soluble and stable rotaxanes that exhibit some desirable characteristics, I don’t think these rotaxanes (the ones reported) could be used in broad biomedical applications. They show little retention in the tissues, so I find this application really hard, because they don’t monitor their dyes after two hours of being injected, and you can’t really see what happened before that or how they were distributed in the organs. On the other hand, these compounds do seem to have low toxicities, so maybe future derivatives will in fact be very useful as bioimaging tools.




16 responses

2 05 2012
Ana Victoria

Smith and coworkers describe the synthesis of water-soluble squaraine rotaxanes, their absorption/emission properties, and their chemical stabilities in conditions similar to those of physiological environments. The purpose of doing this is to create future bioimaging markers that can be used in vivo. Each dye presented an absorption band in the region of abot 650 nm and a fluorescence emission with a 20 nm Stokes shift. Chemical and photochemical stability were measured by monitoring the absorbance in water and FBS. It was found that the rotaxanes with four stopper groups were the most stable followed by three and two respectively. This can be explained because the reactive core is less exposed, which makes sense. Photochemical stabilities of these stable rotaxanes showed very little absorbance loss. These results were pretty much expected and the graphs that they present are easy to understand, shows the evidence nicely and confirms their findings. I think that these were the proper experiments. These rotaxanes presented a low affinity for albumin protein, suggestion its potential use as a control tracer to develop probes for in vivo molecular imaging. Because of this, some mice were sacrificed and treated with the dyes. Fluorescence images indicate that they stayed in the urine bladder mostly, even though they exhibited a low retention they will do more future studies regarding this issue. However, I think that because of the low retention, it will be very difficult to find the biomedical application for these rotaxanes, but it is a good start. Maybe their next article will explain better and in more detail their findings in this area to support their hypothesis.

Loru’s synopsis was well written in terms of spelling and grammar as well as the ‘flow’. It was easy to understand and I think that a person with a general knowledge might be able to understand some parts if they put an effort to look some words up in Wikipedia. I think she followed most of the guidelines, she did not use supplementary material for example.

26 09 2012
Erin L. Cole

The SR scaffold can be modified in a variety of ways. This paper focuses on SR with COOH group for amide bond coupling and non-targetted tracer dyes. Other papers have focused on targeted SR such as the Zn-DPA SR which target bacterial infections and cancer.

2 05 2012
Luis Negrón

This is ones of those articles with more than an appropriate content, in this article we can see a significant problematic that face many researchers and how far we can be after solving that main problematic that in this case is the water solubility problem. In this article of Smith, rotaxanes has their VIP PASS with water solubility and their absorption/fluorescent properties are studied with different solubilizing groups. This terminal chains or solubilizing charged groups, play a key role in the stability of the resulting fluorescent squaraine rotaxanes. According with their results, 7-8 are the most stable derivatives of their library. Consequently, these two derivatives were studied as fluorescent tracers in mice. They found that 8 was easily excreted from the mouse body.

It’s interesting how the stability of the compound changes with the terminal group, because it’s seems that the proximity of the positive charge have an effect in the stability of the system. Even that the length of the chain looks similar, the ability to organize or arrange in water changes significantly. It will be interesting if the less stable compounds can be more stable by increasing the chain length or flexibility.

I’m not a big expert in fluorescence maps, but it seems that 7-8 are more selective to the bladder instead of ICG that is less organized in the organs. It will be interesting to know why these types of compounds don’t accumulate in the liver. Also, is accumulation of these kind of compound can quench the fluorescence or kill the living mice. It’s interesting to see how some positive compounds can accumulate and forms salts that can damage tissue or affect the health of a living system. So, the easy excretion of 8 is a big advantage of these systems for future applications with this approach.

About Loru’s synopsis, it’s seems that she is growing a lot in her development of scientific writing. The synopsis was well written by exposing the main results and their personal opinion was very smart. I recommend to write the impact of that results instead of just presenting them, sometimes we do some summary in conclusions, but for the reader it will helps if the writer expose the result and explained in terms of the importance of that result. About the picture, it was very interesting, very creative, but the ring was little confusing.

3 05 2012
Diana S.

For those who were not here back than, Bradley Smith was a RISE guest at the UPR a few years ago (2009, perhaps), and in his seminar he presented initial steps of this work, which began with the arrival at the lab of a post-doc with expertise in squarine dyes. Since the discovery that these labile dyes could be efficiently protected using rotaxanes, Smith has published several times on the development of squarine rotaxanes as deep-red imaging tools. The attractiveness of non-invasive bioimaging using wavelengths that can penetrate body tissue is definitely worth the effort.
Through the years, they’ve prepared dyes that quench when encapsulated, and others that don’t; others that light up only in presence of chlorine, or that target bacterial cell walls. Most of them were mostly organic-soluble versions, and now we see the water-soluble generation. If anyone is really interested in the full story (it a really good story), you can look up DOI: 10.1039/b911064j.

In this Org. Biomol. Chem. paper they announce that they managed to get the water-soluble derivatives (well, at least to 20 uM) using two dendritic arms, each ending in four water-solubilizing functionalities (ammonium and carboxylate ions). They also think that are responsible for the quick physiological clearance of the dye, due to hydration of the dye periphery. It sounds interesting, but you’d need to look at other systems as well to support such a general conclusion… Still, the data speaks clearly: the compounds last only minutes in the body of the mice.
Dialysis of the dye against albumin shows little affinity, which leads them to conclude that this is a non-selective tracer. I’d think you’d need to test the compound against other proteins/biomolecules to make such a general conclusion, but again, the experiment conducted in the mice shows that its is not selecting any particular organ, nor is it showing affinity/retention in the bloodstream, etc. so I guess that the conclusion is correct…
I was surprised (and a little disappointed) that they didn’t show any live mice imaging: they were so close…
So, what is the use of a non-selective tracer? Blood stream imaging?? I’m a bit curious about this, since they don’t really talk about the use of these imaging tools, and its not really something I’ve heard before. Could’ve added a sentence on this. Other than that the paper flows nicely, figures are attractive, etc.
The synopsis is a synopsis, covers the main points, etc. It takes quite a bit to be brief and meaningful, and I think that it could’ve been better achieved. For such a conceptually simple paper, the important part is the critical analysis, which would’ve benefited from a broader knowledge on Smith’s work. The narrative of the synopsis is good, and the picture is creative and of good quality.

3 05 2012

In this article is presented a series of water soluble squaraine fluorescent rotaxanes. In total they synthesized 8 deivatives of the squaraine groups, 1-3 had two stopper groups, 4 and 5 had three and 6-8 had four stopper groups. They did absorbance studies and it showed that the quantity of stopper groups is closely related to the stability of the system. This lead to less stability for the 1-5 derivatives because they showed hydrolytic property. In he case of derivative 6 also showed some decomposition. So they are left with compounds 7 and 8 that with affinity studies showed low affinity for albumin. This gave them the possibility of a non-targeted tracer, therefore they injected their rotaxanes in rats. Via fluorescence intensity maps they could see the presence of the rotaxanes and no selectiveness was observed.
The article in general was great and engaging narrative. They go from the synthesis of the derivatives to the application. What is so important about this article is that this system is water soluble and at least two of their derivative can “survive” an in vivo experiment. What seems to be odd is that their standard dye absorption was taken in CHCl3 as a solvent when we are talking about being in water. But I guess that to have a standard in water you have to go into the derivatives that they already have. There must be a way, any takers on that?
About Loru’s synopsis I think she did a great job explaining the paper and putting her opinion out there. And gave some links for extra info. The picture was very creative I guess. I haven’t seen or read Lord of the Rings….I assume it has to do with ring hehehe.

26 09 2012
Erin L. Cole

The Quantum yield equation takes the refractive index of solvent into account. Tetra methyl squaraine dye is a reliable std with a refractive index of 0.7 in chloroform (another std would have worked also). The main thing about a standard is that the abs/em profiles are close in wavelength to that of the target dye.

26 09 2012
Erin L. Cole

Quantum yield of 0.7 (sorry can’t type)

3 05 2012

The discussion of synthesis of squaraine rotaxane dyes and their photochemical properties along with their biological imaging studies were well described in this article by Bradley Smith et al.
The encapsulation of squaraine dyes inside macrocyclic tetralactams creates squaraine rotaxanes. This encapsulation increase dye stability and also prevents the problem of fluorescence quenching upon self-aggregation. These squaraine rotaxanes can be employed as targeted fluorescent molecular probes for cell microscopy and whole-body imaging of disease models. They used the Leigh-type clipping and ‘click’ reactions in the preparation of water-soluble 8 rotaxanes. By using absorbance studies in water, and in 10% fetal bovine serum they found that the chemical and photochemical stability almost increases with increasing of stopper group’s and size, among these 8 rotaxanes they found two rotaxanes can be used as non-targeted, fluorescent tracers. In the in vivo imaging studies of mice showed that two rotaxanes signals were in the bladder while the reference (ICG) was in the intestines.
To be honestly I did not get the difference and the use of both experiments in vivo and ex vivo.

The synopsis is very good in length. Regarding the figure I can understand it but the arrows giving some wrong information for me, I mean one stoppers squaraine rotaxanes prepared from another less stoppers squaraine rotaxanes, I think in this case ‘greater than symbol’ is appropriate and also fluorescence is missing from figure.

26 09 2012
Erin L. Cole

When doing in vivo imaging it is standard to do an ex vivo study of the bio-distribution of the probe. When looking at an image of a whole animal (live or sacrificed) the depth of the tissue affects the fluorescent detection. So an organ close to the surface looks bright and you might conclude that the majority of the probe is there. However when you do the ex vivo imaging of the organs you might find that an organ at deeper tissue depth actually had more probe, but the fluorescence signal was lowered.

See Nature Chemistry DOI: 10.1038/NCHEM.871

3 05 2012

I will like to start with the first sentence of the second paragraph: “The chemical conversion of hydrophobic fluorescent dyes into water-soluble, non-aggregating compounds is a non-trivial task”. Professor Bradley D. Smith is has been studying cell imaging and program cell death using suparamolecular chemistry as the principal tool. To achieve this goal his research team design and synthesize (both covalent & none-covalent) biocompatible fluorescent molecules with near infrared (IR) emission. They have written a few articles and patented the used of quantum dots as fluorescent probes. In this blog article eight water-soluble squarine rotaxanes are synthesized and the stability and interaction with rats was studied. Here we should pay attention to the new functionalities that imparts water solubility. From the eight compounds, 6-8 are the most interesting structure since they start resembling a small generation of dendrimers. Denrmimers have previously being used in NMR contrast agents.
One of the things learned from the article was about the wavelength window needed to be able to have a good bio-imaging performance. A large Stoke shifts are needed, small Stokes shifts produce “inner filter effect” and in for this family of squaraines it was ~20 nm. Moderate quantum yield (quantum yields of 0.16– 0.24) working at concentrations < 0.014 mM. The stability studies by irradiation of the sample during two hours were clever.

The results regarding the treatment of the rats with dyes change the article perspective. I really enjoy this type of studies. Although the pictures are nauseous, if I ever write a paper with this type of results, I will add the figures only for the final version. Smith and coworkers has previously showed in 2010 the use of squarine rotaxanes for cell imaging in rats. What I never read before is the detail regarding the blue urine. What it means that the squarines are not retained in animal tissue? Is this a negative characteristic of the dye or it is positive because it means it have complete clearance?

I would have made two more compounds 1) with two “large stopper group” as for example R5, in what they call the “Less Stable” and 2) three of the same “large stopper group” (R5) (see Scheme 1). This will give me information on how the small dendrimer is affecting the stability.
I like Lorus’ synopsis very much, it’s way better than mine. It helps me catch those important details needed to appreciate the article better. In contrast I am not a fan of her cartoon. How important was ring in for this article? I think I don’t understand the figure very well.

26 09 2012
Erin L. Cole

The reason for the “blue urine” detail is that the bladder is almost impossible to extract whole from the mouse so Bryan Smith measured the content of SR in the urine. For this application it is a positive that a non-targeted tracer dye is being cleared from the mouse. The failure of ICG is that it binds albumen and therefor is retained.

3 05 2012

In this article Dr. Bradley Smith and colleagues reported on the synthesis of 8 water soluble squaraine rotaxanes that differ in the size of the four stopper groups appended at the ends of the encapsulated squaraine dye. Photochemical properties of the squaraine rotaxanes were evaluated in water showing a narrow deep-red absorption band with Stokes shift of ~20 nm. These compounds have modest quantum yields between 0.16 and 0.24 but monitoring the absorbance maxima in water and 10% FBS show that only compound 7 and 8 were the most stable over time. The trend in stability was rationalize based on the size of the of the stopper groups to reduce the translational mobility of the surrounding macrocycle. However I consider that wise selection of the groups at the end of the stoppers have to be done to also prevent aggregation of the squaraine rotaxane by hydrogel or polymer formation which is what I was expecting to see if they go to much higher concentrations of 6 since it has four guanidinium groups. Since it is a matter of size of the stoppers, I was wondering why they didn’t evaluated a squaraine rotaxane having four positively charged bis(zinc-dipicolylamine) which is the Zn-containing group used at the end of R3 (Scheme 1). I have particular interest on evaluating the water solubility and photochemical properties of this compound, if possible, since this Zn-containing group is part of the structure of their already patented PSVue® which is a different type of system but it is commercially available as a robust probe for detecting apoptosis ( I think it might have to do with the synthetic challenges behind the synthesis of what I am asking because similar stoppers as the one described in R3 (Scheme 1) have been previously used by him through his long history working with squaraine rotaxanes. By the way I think is excellent that Dr, Smith is able to have the direct links to most of his articles so we can quickly go through them learning about his work, thanks Brad! (

To evaluate the potential of compounds 7 and 8 as probes for in vivo molecular imaging, first the authors checked that the brightness of these compounds remains unaffected between the pH ranges from 6 to 10. Also it was confirmed that there is only very weak association between this squaraine rotaxanes and albumin protein which is excellent for their use as non-targeted, fluorescent tracers with deep-red emissions. The experiments using the mice revealed the negligible uptake of these compounds by mammalian tissue but I agreed with Loru in the fact that we need more details about what happens with the compounds before two hours. However the authors left us wanting to know more which is good and it is even better that they seem to be working on a more detailed story for the utility of compound 8. This is a very nice piece of the story on squaraine rotaxanes and their possibilities… I consider that in general the authors made the appropriate control experiments even for those using mice. Regarding the latter what I think is missing will be probably include in their next article describing the use of 8 as whole-body molecular imaging probe on living animals.

Loru’s figure was catchy and interesting, I really like it since it highlights the importance of having a rotaxane to stabilize the squaraine but I think that the novelty of the article is beyond that. Therefore she might have highlighted even more the size of the stoppers which is what holds the ring in the right place. One way to do that could be by having the white/red ovals bigger than “the ring”. Her synopsis was really good, I know she worked hard to polish it so congrats, it was worth it. You manage to keep it simple yet having all the interesting details plus your personal criticism which was good.

3 05 2012
Rocio Roque

In this article the authors describe the synthesis of 8 water-soluble squaraine rotaxanes and their absorption/emission properties. The rotaxanes were synthesized using a Leigh-type clipping reaction that traps the squaraines inside the macrocyclic tetralactams. They found out by absorbance studies, that the stability in water of these rotaxanes is related to the number and size of the stooper groups. Concluding that rotaxanes with four large stoopers attached to the squarine were more stable, followed by three and two groups. Their findings show that these squarine rotaxanes can have various biomedical applications.
The article was not too complicated and I think someone with general knowledge of chemistry would’ve gotten the message (…maybe looking up some of the unknown terms presented in the article). Their findings were clear and precise; their conclusions supported by the presented results. About Loru’s synopsis, it was well organized and she was able to incorporate all the important aspects of the paper. Her picture was nice and attractive!

3 05 2012

This article presents the synthesis of interesting deep red dyes (squaraines) that could be useful for biomedical applications. These are not the first deep red dyes that have been synthetized for these purposes. However, they are definitely interesting as they have useful properties that separate them from the rest, for example, they don’t self-quench. The challenge was constructing a dye that was both stable and water soluble, which is what they accomplish in this article. The authors then proceeded to test their compounds in living rats to see if they could serve as potential imaging tools.

This article is a nice step for this group and reminds me of our own water solubility troubles that still continue to plague us. The narrative of the article was nice although it gets a little dense as it approaches the live rat studies. The need for stable water-soluble squaraines was appropriately described in the introductory part of the article so, it was put in the proper perspective.

Loru’s English is great! Her synopsis was a good summary of the work reported in this article. She followed the guidelines (pretty much all of them in the last paragraph. I like her picture although it takes away the focus of the article away from the water solubility and stability. Some one with a general knowledge of chemistry could in fact understand this synopsis with the aid of Wikipedia.

4 05 2012

In this article they describe the absorption and emission properties of a group of water-soluble squaraine rotaxanes and they test their chemical stabilities in conditions that are analogous to physiological environments. In order to study the chemical and photochemical stability, they monitored the absorption maxima and 10% fetal bovine serum. They observed that the stability was dependent on the total number of large stopper groups attached to the two terminal squaraine nitrogen atoms. They also tested the photochemical stability of 7 and 8 by irradiating with a fluorescent bulb for two hours and saw only a small amount of loss in absorption. In these two rotaxanes, they also observed that the brightness remains the same over a pH range from 6 to 8. They then tested them in mixtures of bovine serum albumin and saw that there was weak dye/albumin association, which possibly made them good for use as non-targeted control tracers. However, when they studied these in mice, they saw that the rotaxanes were excreted to the kidneys within minutes of being administered.

I liked the paper, it was easy to read, the narrative was easy to follow and the language was uncomplicated. I didn’t spot any grammatical errors or anything. I think they effectively explain their main reasons for undergoing this investigation and it was a pretty thorough and complete article. As for Loru’s synopsis, I have to say that it was very well written, she followed the guidelines well and the hyperlinks were nice and she did a good job of analyzing the article. Regarding the picture, I think it looks awesome, and I do love Lord Of the Ring, but it may not have been the most clear rendition of what the paper is about. However, I always appreciate how clever and creative Loru’s synopsis pictures are, she never fails to come up with something out of the box.

26 09 2012
Erin L. Cole

In reply to Loruhama M. Delgado about the tissue retention please see our article on Traumatic Brain Injury in which we used SR8 from this paper to monitor the blood-brain-barrier. | ACS Chem. Neurosci. 2012, 3, 530−537

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