Tracking nanodrugs inside mice

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Toda’s paper addresses a huge challenge in targeted drug delivery: the biodistribution of drugs! It’s a very cool paper.

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Tracking nanodrugs inside mice

Targeted drug delivery is a promising approach to improve the effectiveness of treatments and minimize side effect: using advanced nanocarriers, drugs can be delivered specifically to specific cells, tissues or organs. This maximizes the drug concentration at the desired site, while reducing the amounts sent to healthy areas: a win-win situation. This becomes particular crucial for biological drugs (proteins, RNA, DNA) since they are large, charged molecules that struggle to reach their targets efficiently. A perfect example is the COVID vaccine, where RNA is encapsulated in lipid nanoparticles (LNPs) to protect it and help it enter cells. Another is DNA origami, which can be used to create nanoparticles which are easily modified to target different cells or organs. A critical challenge for the field is to study the distribution of these drugs inside animals, so that the design of nanocarriers can be guided towards maximizing specificity and reducing off-target effect. Unfortunately, current imaging techniques for whole animal bodies (PET, CT, bioluminescence imaging, etc.) have limitations:

• They lack single-cell resolution, making it impossible to see which cells are interacting with the drug

• They have low sensitivity, so they require unrealistically high doses of nanoparticles, leading to unrealistic pharmacokinetics

• They can’t capture low-intensity off-target accumulation, which is essential for identifying potential side effects.

On the other hand, more traditional histology-based methods have subcellular resolution and high sensitivity, but they rely on 2D tissue slices, which are not really suitable for the analysis of a whole animal.

To address these limitations, today’s paper introduces Single Cell Precision Nanocarrier Identification (or just SCP-Nano for short): an imaging pipeline combining advanced tissue clearing, high-resolution fluorescence microscopy and AI for mapping and quantifying the biodistribution of fluorescent nanocarriers in the entire mouse body. SPC-Nano has 3 main components:

1. DISCO whole mouse clearing: This technique allows to clear the whole body of a mouse. The authors optimized it to preserve the fluorescent signal of the nanoparticles, reduce signal loss and minimize tissue damage. This allowed them to achieve single-cell resolution in a whole mouse!

2. Whole body imaging: Light sheet fluorescence microscopy is used to image the entire mouse body. Using the optimized DISCO protocol they developed, they achieved a lateral resolution of ~1–2 μm and an axial resolution of ~6 μm! So yeah, it was enough to see single cell. This provided high-throughput, high-resolution 3D imaging of nanocarriers-labeled cells in situ.

3. Deep learning-based image processing: Did you think we could go too many issues without AI? Of course not. The imaging they used allows very high resolution, and to have a reliable and unbiased quantification to compare different conditions the authors turned to AI. The authors had trouble finding existing methods that were reliable enough: so they just developed their own! They used virtual reality-based annotation to create a dataset to train a deep learning algorithm to identify the nanocarriers. This algorithm is capable of identifying individual cells even in highly crowded areas! Very cool.

The authors then applied this pipeline to a variety of nanocarriers, showcasing its versatility. The first nanocarrier was just simple lipid nanoparticles (LNPs). They studies how the delivery method changed the organ-specificity:

• Intranasal administration: primarily targeted lungs.

• Intravenous injection: favored liver and spleen accumulation.

• Intramuscular injection: reduced liver accumulation, favoring spleen targeting instead.

This shows that intramuscular injection could a better strategy for vaccine delivery, since it increases the targeting of immune organs.
Talking about vaccines, their second carriers of choice was SARS-CoV-2 spike mRNA LNPs, very similar to the COVID vaccine: most techniques struggle to image the low concentrations needed for vaccines, while this technique handles them with no problem. The authors actually show that some off-target mRNA accumulates in the heart tissues, and proteomic analyses showed changes in immune-related proteins. This aligns with clinical reports of myocarditis and pericarditis following mRNA vaccination, and it shows the power of this technique to detect off-target effects.

The last two nanocarriers they showed are more speculative. The first one is a DNA origami structure: they showed that a nanorod conjugated with antibody fragments targeting immune cells efficiently accumulate in the target cells, demonstrating the potential of DNA origami for precision drug delivery. Last but not least, they also tested Adeno-Associated Virus (AAV), which are promising carriers for gene therapy. They tested 2 types, and they both successfully targeted neurons: one of them though, unexpectedly, also targeted adipose tissue! Which could be a problem for some AAV-based gene therapies, but maybe good for therapies targeting, for example, obesity-related disorders?

To finish, this paper is a pretty big step forward for the field of targeted drug delivery: it can help with nanocarrier optimization, because you can literally see where your drug is going, and figure out if you have off-targets. It also allows to use clinically relevant drug levels, which reduces high-dose animal studies, making nanocarrier screening more precise and ethical (I can’t wait for the day we won’t be needing animal studies anymore!). And it could even be adapted for use in human tissue imaging, enabling tailor treatments to individual patients!

But of course, this method is not without limitations, which are also pointed out by the authors:

• The method is quite complicated, both on the experimental and on the data analysis side

• It’s not a live animal technique (like PET or bioluminescence imaging), so can’t track real-time nanocarrier movement

• It only works for carriers or therapeutics can be fluorescently labeled

But even with these limitations, I think this was a great work! So go read it for yourself here.

As always, thanks for reading! And don’t forget to share it if you enjoyed it!

More room:

• Pushing DNA nanotech into mechanobiology: Mechanobiology studies how molecular forces influence cell behavior and tissue development, and I find it pretty cool. This review highlights the role of DNA-based nanodevices in molecular diagnostics, organ engineering, and tissue regeneration, outlining current progress, challenges, and future directions in this rapidly evolving field. I know what I am going to read the next days!

• Fluorescent rotor amino acids? This study introduces fluorescent molecular rotor amino acids (FMR-AAs), a new class of genetically encoded fluorogenic amino acids inspired by native fluorescent protein fluorophores. By incorporating FMR-AAs into non-fluorescent proteins via an expanded genetic code, researchers can transform them into artificial fluorescent proteins. These engineered proteins function as biosensors to monitor protein–protein interactions and conformational changes in living cells. I am not sure I follow everything, but it looks cool!

• Sticking DNA origami on silicon: Do you want to use your DNA origami to make chips? Well, then this study could help you out (or maybe not). This study examines how different silicon oxide (SiOx) surfaces affect the self-assembly of DNA origami lattices, which have potential applications in molecular lithography. The authors tested different conditions and techniques, and these findings highlight the importance of optimizing SiOx surface properties (particularly low roughness and high oxide density) to achieve high-quality DNA origami lattice assembly for nanofabrication applications.

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