Matrix-bound Vesicles as Tissue-specific Extracellular Vesicle Models for Biosensor Development.

Introduction:: Heart disease remains the leading cause of death in Puerto Rico and the US. There are limited options for monitoring cardiovascular diseases, and current monitoring methods involve high radiation exposure, which has a grade of invasiveness and other side effects. Endothelial extracellular vesicles (endo-EVs, CD144+ -endothelial cell marker-) are new biomarkers associated with the prognostic and diagnostic of cardiovascular diseases. Endo-EVs are found in body fluids in combination with other EVs and biomolecules as a heterogenous population hampering its use as an EV model. We need an endo-EV model to develop a selective biosensor. Matrix-bound vesicles (MBVs), EVs embedded within the decellularized extracellular matrix (dECM), are a new class of tissue-specific EVs. Our hypothesis is that MBVs isolated from endothelial tissues (endo-MBVs) will carry the CD144+ marker and function as a physiologically relevant model for developing EV biosensors. We seek to build an EV biosensor for the selective detection of endo-EVs.

Materials and Methods:: To accomplish this, we first must i) derive endo-dECM from porcine endothelium, ii) isolate endo-MBVs, and iii) characterize them as CD144+ to use them as an EV model for biosensor construction. We have developed a method based on sodium deoxycholate to decellularize endothelium–ECM. The MBVs were isolated using dECM solubilization via KCl solution. Then, the MBVs were quantified and characterized using methods validated for exosomes (i.e., nano-tracking analysis -NTA-, Exocet, microRNA staining). An immunoblotting technique (dot blot) was used to detect CD144 in the isolated end-MBVs. Finally, treated screen-printed electrodes were incubated with CD144+ MBVs, and electrochemical characterization was performed.

Results, Conclusions, and Discussions::
The decellularization protocol developed yielded 30-50 mg of endo-dECM. Using overnight solubilization, the KCl solution could extract the MBVs embedded within the endo-dECM, which were further purified using a polymer precipitation kit validated for EV isolation. We characterized the size of the MBVs particles to be approximately 130 nm using nanoparticle tracking analysis (NTA). The MBV concentration was quantified as 10^8 EVs/uL. The immunoblotting test identified the endothelial protein marker CD144 in the MBVs. Electrochemical characterization successfully showed the detection of CD144+ MBVs on screen-printed electrodes.




Summary/Conclusion




This project provides evidence for the isolation of CD144+ MBVs that can be used as a target model for engineering an endo-EV biosensor. The outcome of this research has a broader impact, as it supports the use of MBVs as tissue-specific EV models for developing biosensors and other related technologies.




Acknowledgements (Optional): : NIH R13 No. 1R13EB030941-01 in association with the 2023 TERMIS-AM conference in Boston, NSF Award No. OIA-1849243, NSF Engineering Research Center for Cell Manufacturing Technologies No. EEC-1648035, NIH-3SC1GM131967-03S1

References (Optional): : 1. Bordanaba-Florit, G., Royo, F., Kruglik, S.G. et al. Using single-vesicle technologies to unravel the heterogeneity of extracellular vesicles. Nat Protoc16, 3163–3185 (2021). https://doi.org/10.1038/s41596-021-00551-z

2. A. Desgeorges, J. Hollerweger, T. Lassacher, E. Rohde, C. Helmbrecht, M. Gimona, Differential fluorescence nanoparticle tracking analysis for enumeration of the extracellular vesicle content in mixed particulate solutions, Methods, Volume 177, 2020, 67-73, https://doi.org/10.1016/j.ymeth.2020.02.006. (https://www.sciencedirect.com/science/article/pii/S1046202319302646)

3. Martín-Gracia, B., Martín-Barreiro, A., Cuestas-Ayllón, C., Grazú, V., Line, A., Llorente, A., M. de la Fuente, J., & Moros, M. (2020). Nanoparticle-based biosensors for detection of extracellular vesicles in liquid biopsies. Journal of Materials Chemistry B, 8(31), 6710–6738. https://doi.org/10.1039/d0tb00861c

4. Turner NJ, Quijano LM, Hussey GS, Jiang P, Badylak SF. Matrix-bound nanovesicles have tissue-specific characteristics that suggest a regulatory role. Tissue Engineering Part A. 2022;28(21-22):879-892. doi:10.1089/ten.tea.2022.0091