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Tracking Exosomes by Bioluminescence Imaging

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Exosomes are nanosized lipid bilayer vesicles, released by almost all cell types, capable of carrying biologically active molecules such as proteins, lipids, RNA, and DNA. Research has found that exosomes are natural carriers essential in many physiological and pathological processes. The amount and composition of exosomes in the fluids of human organisms can be used as biomarkers to diagnose diseases and monitor the effectiveness of treatments. Exosomes are also expected to be used as natural carriers for therapeutic agents and drug delivery. However, successfully using exosomes in clinical practice requires understanding their distribution in the organism and their interaction with target cells.

Figure 1. Tracking exosomes using signaling molecules (pHluorin, NLuc, Antares2, GFP, and GLuc).Figure 1. Genetic engineering for Exosome labeling. (Liu Q, et al., 2022)

Bioluminescence imaging (BLI) is widely used in preclinical research and has been used to analyze exosome release and uptake processes, as well as in vivo model systems to visualize exosome distribution.

What is BLI Technology?

BLI is the most widely used in vivo imaging technology, using light generated by natural biological processes (i.e., luciferase-substrate reactions) and an ultrasensitive camera for signal detection, enabling visualization of molecular and cellular processes under normal and pathological conditions. The main advantage of BLI is the high signal-to-noise ratio (SNR), as mammalian tissues are usually luciferase-free and do not emit light. Depending on the type of luciferase, the SNR can reach about 102 -107. BLI does not require an excitation source for luminescence but emits bioluminescence through the reaction of the respective substrate with ATP and Mg2+ or O2 alone. The high sensitivity and low background signal quality of BLI make the luciferase gene a good choice for tracking exosomes in vivo.

What are the Commonly Used Luciferase Systems for BLI?

Commonly used luciferase genes for BLI are Firefly luciferase (Fluc), Gaussia luciferase (Gluc), or Renilla luciferase (Rluc). Luciferase from terrestrial organisms uses d-luciferin as a substrate and ATP, Mg2+, and O2 as cofactors, while luciferase from marine organisms uses a luminalin analog as a substrate. The new luciferase NanoLuc (460 nm, blue light) is of particular interest. It is isolated from the deep-sea shrimp Oplophorus gracilirostris. This bioluminescent platform has a smaller size and higher luminescence levels compared to other known systems.

Figure 2. Commonly used bioluminescent labels and their maximum emission wavelengths.Figure 2. Overview of commonly used bioluminescent labels. (Boudna M, et al., 2024)

How can BLI be used to Track Exosomes?

BLI of exosomes first requires labeling these extracellular vesicles with luciferase reporter proteins. The luciferase gene is fused to different membrane protein genes (e.g., ALIX, TSG101, four transmembrane proteins such as CD9, CD63, CD81, etc.) to form an engineered vector plasmid. Then, the plasmid is transfected into target cells and cultured for an appropriate period to release exosomes carrying the appropriate reporter proteins. Before imaging, appropriate substrates (RLuc/GLuc-Coelenterazine, FLuc- d -fluorescein) were injected into the organisms to observe the biodistribution of exosomes.

Application Examples of Using BLI to Track Exosomes

Scientists have improved specificity and enhanced exosome-mediated cargo delivery to pancreatic β-cells by modifying the exosome surface with a pancreas-specific p88 peptide. Lactamyxin is a membrane-associated protein that fuses with the p88 peptide. In addition, donor 293T cells were transfected with a plasmid encoding Gaussian luciferase (Gluc). Research has shown that signals from non-targeted exosomes are found primarily in the lungs and spleen, whereas bioluminescent signals from targeted EVs (containing p88 peptides) are observed primarily in the lungs, spleen, and pancreas.

Figure 3. Schematic imaging of organs in mice injected intravenously with luciferase.Figure 3. Representative images of the organs from the Balb/cJ mice received intravenous injections of PBS, NP-gLuc- or p88-gLuc-EVs. (Komuro H, et al., 2021)

To research whether tumor-derived exosomes can preferentially target their parent cells, researchers used renal luciferase (RLuc) to label thyroid cancer cell-derived exosomes and monitor their homologous targeting ability. Bioluminescence imaging showed that exosomes targeted homologous tumors in mice within 30 minutes of intravenous injection. In addition, the subcellular distribution of exosomes in excised tumors was visualized by immunofluorescence. The in vitro results further confirmed that most exosomes co-localized with tumor cells.

Figure 4. EV-CAL62/Rluc Representative ex vivo bioluminescence imaging of excised tumors.Figure 4. Ex vivo and Subcellular visualization of EV-CAL62/Rluc in tumors. (Gangadaran P, et al., 2018)

Why Choose BLI for Exosome Tracking?

  • Sensitivity - BLI offers high sensitivity, allowing the detection of small amounts of exosomes.
  • Real-time monitoring - BLI's ability to enable dynamic tracking of exosomes in an organism provides valuable insights into their biodistribution and targeting efficiency.
  • Quantitative Data - BLI allows quantification of exosome concentrations, which is valuable for assessing the pharmacokinetics and bioavailability of exosome-based therapies.

Our Products

Exosome tracking based on bioluminescence imaging has been successfully used in cancer occurrence and treatment research. In addition to exosome labeling and tracking services, we also provide a comprehensive range of exosomes from cancer cell lines to help clients more deeply research the potential applications of exosomes in cancer therapy and as drug delivery vehicles.

Cat No. Product Name Source
Exo-CH06 HQExo™ Exosome-MCF-7 Exosome derived from human breast cancer, noninvasive cell line (MCF-7 cell line)
Exo-CH07 HQExo™ Exosome-MDA-MB-231 Exosome derived from human breast cancer, aggressive/invasive/metastatic cell line (MDA-MB-231 cell line)
Exo-CH10 HQExo™ Exosome-HCT116 Exosome derived from human colorectal carcinoma cell line (HCT116 cell line)
Exo-CH04 HQExo™ Exosome-H196 Exosome derived from Human small cell lung cancer cell line (H196 cell line)
Exo-CH09 HQExo™ Exosome-DAUD1 Exosome derived from human burkitt lymphoma cell line (DAUD1 cell line)
Exo-CH16 HQExo™ Exosome-COLO1 Exosome derived from human colon carcinoma (COLO1 cell line)
Explore All Exosomes Isolated from Cancer Cell Lines

If you are interested in our exosome tracking service, please feel free to contact us. We look forward to working with you.

References

  1. Liu Q, et al. Tracking tools of extracellular vesicles for biomedical research. Front Bioeng Biotechnol. 2022. 10: 943712.
  2. Boudna M, et al. Strategies for labeling of exogenous and endogenous extracellular vesicles and their application for in vitro and in vivo functional studies. Cell Commun Signal. 2024. 22(1): 171.
  3. Komuro H, et al. Engineering Extracellular Vesicles to Target Pancreatic Tissue In Vivo. Nanotheranostics. 2021. 5(4): 378-390.
  4. Gangadaran P, et al. New Optical Imaging Reporter-labeled Anaplastic Thyroid Cancer-Derived Extracellular Vesicles as a Platform for In Vivo Tumor Targeting in a Mouse Model. Sci Rep. 2018. 8(1): 13509.
  5. Aimaletdinov AM, Gomzikova MO. Tracking of Extracellular Vesicles' Biodistribution: New Methods and Approaches. International Journal of Molecular Sciences. 2022. 23(19): 11312.
  6. Jiang A, et al. In Vivo Imaging for the Visualization of Extracellular Vesicle-Based Tumor Therapy. ChemistryOpen. 2022. 11(9): e202200124.

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