By Yanira Rodríguez Valdéz
The article by Swaminathan et al. studies the Förster Resonance Energy Transfer (FRET) between two dyes encapsulated in self-assembled nanoparticles made from amphiphilic polymers. The encapsulation of hydrophobic chromophores such as borondipyrromethene (BODIPY) and anthracene inside the hydrophobic interior of these nanoparticles allows their study in aqueous media. In close proximity (inside the same host), BODIPY and anthracene undergo FRET (anthracene = donor, BODIPY = acceptor). The authors observed that mixing nanoparticles loaded with anthracene and nanoparticles loaded with BODIPY has the same effect as loading nanoparticles with both anthracene and BODIPY, which suggests guests being exchanged when nanoparticles are mixed and that they are being captured by the same nanoparticle(s). These studies are performed both in aqueous media and in vitro (HeLa cells), which confirms that the guests can also be exchanged intracellularly.
Two mechanisms for guest transfer between hosts are proposed: 1) guests escape a host and subsequently are captured by another or 2) collision between hosts leads to exchange of guests. They indicate that because of the poor aqueous solubility of the fluorophore guests, mechanism 2 is more likely, but the authors do not perform any experiments to confirm this. They also state that 10-fold dilution with PBS does not affect energy-transfer efficiency. This is not expected if mechanism 2 is at play. Dilution would lead to less frequent collisions between hosts, and therefore, to less guest exchange, and less energy-transfer. The energy-transfer observed could be explained by diffusion, which is more akin to mechanism 1. Poor solubility might not be an issue, because as long as a small amount is soluble, escapes the host and is further entrapped by a host carrying the opposite guest, energy-transfer can still occur.
The authors attempt to determine where the supramolecular assemblies are localized intracellularly by incubating HeLa cells with two sets of nanoparticles, loaded separately with anthracene and BODIPY, and either chlorpromazine or genistein, which inhibit either clathrin- or caveolae-mediated endocytosis, respectively. The results indicated that intracellular fluorescence was reduced to a greater extent by the addition of chlorpromazine, which suggests that the particles are predominantly internalized by clathrin-mediated endocytosis. However, the authors do not perform further experiments that show the particles localized in endosomes and/or lysosomes, which would result from clathrin-mediated endocytosis. They seem to be attempting to rule out any other methods of internalization, but given that chlorpromazine only reduced the intracellular fluorescence to 59%, other methods of internalization could still be taking place.
Swaminathan, 2014. Intracellular Guest Exchange between Dynamic Supramolecular Hosts
This article as a proof of concept seems ok, yet Yanira’s comment gives a great perspective on the article itself and how it is designed. She also brings to the table on what I dare to call the bold claim by the authors of collision vs. diffusion. Although each claim haves its validity as a possibility or debunked somehow; by looking at other systems and our own, diffusion seems the most reasonable way to explain the phenomenon of re-mixing. Experimentation on system leakage would have given the authors a starting point on what could be happening. Either way, in the overall narrative of the article they seem to not bring this up again. Maybe it was just a question of “reviewer #3” that they needed to answer. Still, Yanira’s discussion seems to be taking in the essence of the paper with a good critique. The figure itself, maybe with a better space management would be more appealing, yet conveys what the authors missed on their TOC figure.
The synopsis presented here is a great summary of the paper. It’s interesting to see the two mechanisms for guest transfer between hosts: 1) guests escaping host 2) collision between hosts and how it enhances the fluorescence of the system.
In the figure, the FRET mechanism is presented and I think that’s one of the most important discoveries of the research. By incubating the HeLa cells just with anthracene or BODIPY the investigators barely saw any fluorescence, but then, due to the FRET process between the two molecules the fluorescence is enhanced. In general, this study is important in order to understand the different interactions that two molecules can have when they are inside a cell which can allow better transport to the extracellular space.
Rating (Synopsis): 5/5
Rating (Figure): 5/5
Yanira’s synopsis is very detailed and critical. It shows attention to details without being excessive. Personally, I loved how her critiques were developed, specially in the last paragraph. I also found interesting what she said about the guest transfer hypotheses. As she explained it, it seems very logical. That made me wonder, could it be possible that the guests are transferred to the host in a combination of both collision and diffusion? Obviously one would play a mayor role in the transfer but, could both occur at the same time? (No need to answer, those are just random questions that came to mind with your critique.) Finally, regarding your image, it was very appealing and comfortable. The only issue it has is the contrast of the fluorescence in the first two microscopy images, but that would be an issue related to the authors/journal.