Tracking Exosomes by Fluorescence Imaging

Exosomes range in size from 30-150 nm and serve many biological functions as nanocarriers for intercellular communication. They contain a variety of biomolecular cargoes including proteins, nucleic acids, and carbohydrates that regulate processes such as homeostasis, immune responses, and angiogenesis under physiological and pathological conditions. In addition, exosomes are promising delivery vectors due to their unique ability to migrate, target, and selectively internalize into specific cells. Thus, exosomes can serve as an alternative to cell-based therapeutics. However, the challenge in translating exosome therapy into the clinic is that less is known about the behavior of these endogenous vesicles in vivo.

Figure 1. Exosome information transfer process and application. (Huang T, Deng CX, 2019)

In vivo tracking of exosomes could provide essential knowledge about their biodistribution, migratory capacity, toxicity, biological roles, communication capabilities, and action mechanisms. Various imaging strategies have been adopted to track exosomes in vivo, including bioluminescence, fluorescence, radiolabeling, and tomography techniques. Among them, fluorescence imaging (FLI) has been widely used because of its non-invasive, low-cost, and technology-friendly.

What is FLI Technology?

With the development of science and technology, optical technology, especially FLI technology, has become a major tool for exploring exosome biogenesis and its action mechanism. FLI is an analytical strategy widely used in biology, pharmacy, and medicine. In recent years, FLI technology has been reported to be used for exosome tracking and imaging due to the simplicity of fluorescence microscopy operation, which can provide information in real time and non-invasively. Numerous studies have tracked labeled exosomes by fluorescence imaging, where the signal can remain constant or change with the environment.

What are the Labelings Used in FLI?

FLI involves the use of endogenous or exogenous molecules or materials that emit light when activated by an external light source. Fluorescent dyes and fluorescent proteins are commonly used as indicators in FLI.

• Fluorescent dyes are the most commonly used materials for exosome labeling, tracking, and imaging. A variety of commercial dyes have been developed including carbocyanine dyes, PKH dyes, and azodibenzylcyclooctyl (ADIBO) dyes.
• Fluorescent proteins (e.g., GFP and RFP), often referred to as reporter proteins, emit fluorescent signals at specific wavelengths of excitation light. They are fused to molecules on the surface or inside the exosome to construct fusion proteins. Therefore, monitoring the fluorescent signals emitted by the fusion proteins means tracking the labeled exosomes.

Fluorescent proteins are mostly introduced into the parental cells-indirectly labeled, and lipophilic dyes are incorporated directly into exosomes. Labeling exosomes with lipophilic dyes mainly provides insights into their biodistribution and uptake. Labeling exosomes with fluorescent proteins reveals the biological mechanisms of exosome biogenesis, secretion, and communication in cells.

Application Example of Using FLI to Track Exosomes

Researchers used lipophilic carbocyanine DiOC18(7) (DiR) to label exosomes from cancer cells (4T1, PC3, and MCF-7), injected 4T1 tumor-bearing mice, and later imaged with a combination of two-dimensional (2D) optical and three-dimensional (3D) optical tomography. It assessed the adriamycin delivery effect of exosomes. In vivo imaging showed that the majority of intravenously injected exosomes accumulated in the liver and spleen 1 hour after administration, with no significant changes at 24 hours, and exhibited rapid clearance and low accumulation in tumors. Intratumorally injected exosomes showed a higher retention rate in tumor tissue than liposomes.

Figure 2. Intratumoral injection of DIR-labeled exosomes and liposomes. (Smyth T, et al., 2015)

Another research used GFP-expressing EL4 lymphoma tumor cells fused to a palmitoylated (palmGFP) reporter sequence for indirect labeling of exosomes. Researchers used multiphoton in vivo microscopy to visualize and track the uptake and exchange of tumor-derived exosomes between cell populations. The highest intensity of exosome secretion was found in the periphery of the tumor, while the center of the tumor had lower levels of exosome secretion. Time-lapse imaging showed that larger exosomes stayed near the tumor tissue longer than smaller exosomes. Exosomes were found to be attached to or internalized by a motile, non-visualized cell population based on the imaging results.

Figure 3. In vivo imaging of IV-administered exosomes. (Lai CP, et al., 2014)

Why Choose FLI for Exosome Tracking?

• High Sensitivity - Fluorescence imaging allows for the detection of exosomes at very low concentrations due to the high sensitivity of fluorescence signals.
• Multiplexing Capabilities - Different fluorescent dyes can be used to label exosomes, facilitating the tracking of multiple populations simultaneously within the same sample.
• Real-time Visualization - It enables real-time visualization and tracking of exosomes in live cells or tissues, providing dynamic insights into their behavior and interactions.
• Quantitative Analysis - Fluorescence imaging allows for quantitative analysis of exosome uptake, distribution, and kinetics, providing valuable data for research.
• Wide Range of Fluorophores - There is a wide range of fluorophores available, offering flexibility in choosing dyes based on parameters like excitation/emission spectra, stability, and brightness.

Our Products

In addition to FLI-based exosome tracking services, we offer a range of high-quality fluorescent exosomes/microvesicles to help clients explore the potential applications of exosomes in anti-cancer research and regeneration.

Based on comprehensive knowledge of exosomes, Creative Biostructure offers a one-stop shop to satisfy clients' requirements for exosome research, including exosome isolation, characterization, labeling, tracking, targeting, and functional analysis. Please contact us for more information.

References

1. Huang T, Deng CX. Current Progresses of Exosomes as Cancer Diagnostic and Prognostic Biomarkers. Int J Biol Sci. 2019. 15(1): 1-11.
2. Smyth T, et al. Biodistribution and delivery efficiency of unmodified tumor-derived exosomes. J Control Release. 2015. 199: 145-155.
3. Lai CP, et al. Dynamic biodistribution of extracellular vesicles in vivo using a multimodal imaging reporter. ACS Nano. 2014. 8(1): 483-494.
4. Liu Q, et al. Tracking tools of extracellular vesicles for biomedical research. Front Bioeng Biotechnol. 2022. 10: 943712.
5. Chen Y, et al. Fluorescence Tracking of Small Extracellular Vesicles In Vivo. Pharmaceutics. 2023. 15(9): 2297.