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Nano and Micro Technologies

Super Resolution Imaging of Liposomes Using Lipid-Retention Expansion Microscopy

(K-418) Super Resolution Imaging of Liposomes Using Lipid-Retention Expansion Microscopy

Friday, October 13, 2023

3:30 PM – 4:30 PM PDT

Location: Exhibit Hall - Row K - Poster # 418

Presenting Author(s)

Abigail Grace Thomas

Student
University of Oklahoma
Grand Prairie, Texas, United States

Primary Investigator(s)

SW

Stefan Wilhelm

Associate Professor
University of Oklahoma, United States

Introduction::

We explored three-dimensional (3D) super-resolution microscopy of model cancer-associated cells with fluorescent imaging of liposomes to study their intracellular spatiotemporal distribution. A better understanding of the intracellular nanoparticle spatiotemporal distributions is required to use nanomedicines safely and effectively. While traditional light microscopy does not allow us to achieve a high enough spatial resolution for visualization, electron microscopy techniques do not allow for the 3D visualization of the intracellular liposomes. In addition, expansion microscopy (ExM) techniques, such as protein-retention expansion microscopy or panExM, use lipid clearing, which is not compatible for the expansion and visualization of liposomes inside cells. Thus, we adapted a recently published ExM protocol called Magnify [1], a comprehensive biomolecule retention method that anchors cell samples to a swellable acrylamide hydrogel to expand them up to 11x their original size for super-resolution imaging. We used this three-dimensional (3D) super-resolution microscopy technique to image the uptake and intracellular distribution of liposomes with visualization of both the stained lipid and its cargo, in both adherent and non-adherent cell lines.

Materials and Methods::

Cultured RAW 264.7 murine leukemia-associated macrophages and THP-1 human leukemia-associated monocytes were used as model adherent and non-adherent immune cell lines, respectively, to study the effects of differentiation on liposome intracellular distribution. Cells were treated with fluorescent TRITC dextran (red)-encapsulated liposomes stained with the lipophilic dye DiO (green) for 24 hours. The cells were then expanded using an ExM process known as Magnify [1], which involves anchoring the biomolecules in cells to an acrylamide-based hydrogel that is swellable in water, resulting in an up to 11x expansion (increase in spatial resolution). To visualize the entire proteome, Magnify-processed cells were stained with AF405-NHS ester dye. The resulting cells were imaged on a standard inverted confocal laser scanning microscope, allowing fluorescent imaging of the liposomes and their cargo. We studied the interactions between dextran-encapsulated liposomes and human THP-1 monocytes before and after their differentiation into macrophages.

Results, Conclusions, and Discussions::

This unique super-resolution imaging approach enables the visualization of organic nanomaterials and intracellular features and compartments with electron microscopy-like spatial resolution in 3D. In Figure 1

we demonstrate that our method allows for the visualization of TRITC dextran encapsulated DiO-liposomes within individual intracellular vesicles, as seen by the colocalized red and green fluorescent signal, and the demonstrated expansion as compared to Figure 2. It also opens up the possibilities to examine nano-bio interactions on non-adherent cell lines, revealing an opportunity to affect fields such as immunoengineering, where non-adherent cell studies are highly relevant. We present the three-dimensional super-resolution imaging of intracellular nanoparticle distributions. Our unique method has the potential to transform the understanding of nanoparticle-immune cell interactions to empower safe and effective nanomedicine applications that require a precise spatiotemporal understanding of intracellular nanoparticle fate.

Acknowledgements (Optional): :

References (Optional): : 1. Klimas, A. et al. Magnify is a universal molecular anchoring strategy for expansion microscopy. Nat. Biotechnol. (2023) doi:10.1038/s41587-022-01546-1.