Tracking Exosomes by Nuclear Imaging

Exosomes are cell-secreted nanovesicles with a lipid bilayer structure that hold great promise as natural therapeutic agents and drug delivery vehicles. For exosomes to be effectively used as therapeutic or drug deployment frameworks in bioscience applications, it is necessary to determine their circulatory dynamics and biodistribution profiles, as well as their targeting potency to precise cellular or tissue and uptake pathways. A variety of imaging techniques have been used for non-invasive in vivo tracking of exosomes, with nuclear imaging standing out in various preclinical research due to its high sensitivity, depth of penetration, and accurate quantification.

Figure 1. Schematic of extracellular vesicle tracking by nuclear imaging. (Jiang A, et al., 2022)

What is Nuclear Imaging?

Nuclear imaging is at the forefront of molecular imaging modalities, including SPECT and PET, and uses radionuclides that emit gamma or positrons as imaging probes and is a highly sensitive technique. Its main advantages over other imaging modalities are high sensitivity, unlimited depth of penetration, accurate quantification, and the ability to typically detect probes in the picomolar to the nanomolar range, making it suitable for non-invasive tracking of exosomes in living animals. These systems, although originally developed for clinical use, have been scaled down to provide high-resolution imaging in small animals, which enhances the translational potential of preclinical research in animal models.

• SPECT imaging is based on the detection of single photons emitted by gamma-emitting radionuclides with energies in the range of 30 to 300 keV and half-lives ranging from hours to days.
• PET is based on the detection of two simultaneous high-energy photons produced by the decay of a positron-emitting radioisotope. The emitted positron annihilates with a nearby electron in the tissue and produces a pair of 511 keV photons traveling in opposite directions, providing a higher-resolution image with higher sensitivity than SPECT.

What are the Labeling Methods Used for Nuclear Imaging to Track Exosomes?

Several strategies have been used to label exosomes with gamma or positron-emitting radionuclides for SPECT and PET applications. The widely used methods fall into three categories:

Figure 2. Three examples of radiolabeling of extracellular vesicles. (Almeida S, et al., 2020)

• Covalent Binding

Unmodified exosomes contain carboxy-terminal phospholipids or transmembrane proteins on their surface that covalently bind to imaging probes. This labeling strategy is based on the formation of stable covalent bonds between naturally reactive functional groups and probes conjugated to chemical groups that react with specific portions of the exosome surface. This method avoids membrane rupture or denaturation of surface proteins, and covalently labeled exosomes are less likely to dissociate due to the strength and stability of these chemical bonds.

• Encapsulation or Intraluminal Radiolabeling

Radionuclides can be encapsulated and captured into the lumen of exosomes and modifications of the exosome surface can be avoided. This method is based on passive diffusion of a hydrophobic probe that spontaneously crosses the membrane and enters the vesicle lumen, or by an active loading strategy.

• Membrane Radiolabeling

Another strategy is the surface functionalization of exosomes using bifunctional chelators (BFCs), which contain reactive amines, thiols, or carboxyl groups for covalent attachment to the exosome surface and a metal-binding portion for radionuclide sequestration. The method is based on click chemistry and provides high yields of labeled exosomes under simple and mild reaction conditions.

Innovations in Nuclear Imaging for Exosome Tracking

Limitations of current imaging tools have hindered a better understanding of the in vivo behavior of extracellular vesicles (EVs). In addition, chemical labeling carries the risk of altering EV membrane characteristics.¹⁹F-MRI is a safe bioimaging technique that provides selective images of exogenous probes. The researchers demonstrated the first example of a fluorinated EV containing PERFECTA, a branched molecule with 36 magnetically equivalent ¹⁹F atoms. PERFECTA-containing EVs are produced naturally by injecting PERFECTA emulsion into cells. PERFECTA-EVs retained the physicochemical characteristics and morphology of native EVs, exhibiting strong 19F-NMR signals and excellent ¹⁹F relaxation times. The in vivo ¹⁹F-MRI and tumor-targeting ability of stem cell-derived PERFECTA-EV is also demonstrated. Scientists suggest PERFECTA-EV as a promising mixture for imaging EV biodistribution and transport throughout the body.

Figure 3. Flowchart of in vivo19 F-MRI imaging of mice bearing HeLa tumors. (Sancho-Albero M, et al., 2023)

Scientists have explored two novel methods of exosome tracking using ¹¹¹In³⁺ as a radioisotope. One method involves an oxine-like radioisotope encapsulation approach that uses tropinone as an ion carrier for ¹¹¹In³⁺ shuttling into the exosome lumen. The second method covalently attaches DTPA-anhydride, a bifunctional chelator, to the surface of the exosome, endowing it with the ability to stably bind ¹¹¹In³⁺. The researchers evaluated the radiolabeling efficiency and stability of the two methods and studied the biodistribution of melanoma-derived exosomes in immunocompetent and immunodeficient melanoma mice to research the effect of the mouse immune system on the biodistribution of exosomes.

Figure 4. Whole-body SPECT/CT image of membrane-labeled B16F10 exosomes in C57Bl/6 mice bearing melanoma. (Faruqu FN, et al., 2019)

What are the Advantages of Tracking Exosomes by Nuclear Imaging?

• High Resolution - Nuclear imaging allows researchers to track exosomes with a higher degree of resolution, compared to other imaging techniques.
• Quantitative Analysis - This method visualizes exosomes and provides quantitative information, such as concentration, which can be important in various research studies.
• Deep Tissue Penetration - One significant advantage of nuclear imaging techniques like PET and SPECT is that they have higher tissue penetration. This allows for imaging and tracking of exosomes in deep tissues which can be crucial in in vivo studies.
• Specificity - Nuclear imaging techniques allow for specific labeling and detection of exosomes, thereby reducing the chances of false-positive results.

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References

1. Jiang A, et al. In Vivo Imaging for the Visualization of Extracellular Vesicle-Based Tumor Therapy. ChemistryOpen. 2022. 11(9): e202200124.
2. Almeida S, et al. In Vivo Tracking of Extracellular Vesicles by Nuclear Imaging: Advances in Radiolabeling Strategies. Int J Mol Sci. 2020. 21(24): 9443.
3. Sancho-Albero M, et al. Superfluorinated Extracellular Vesicles for In Vivo Imaging by 19F-MRI. ACS Appl Mater Interfaces. 2023. 15 (7): 8974-8985
4. Faruqu FN, et al. Membrane Radiolabelling of Exosomes for Comparative Biodistribution Analysis in Immunocompetent and Immunodeficient Mice - A Novel and Universal Approach. Theranostics. 2019. 9(6): 1666-1682.
5. Ashique S, Anand K. Radiolabelled Extracellular Vesicles as Imaging Modalities for Precise Targeted Drug Delivery. Pharmaceutics. 2023. 15(5): 1426.