#4838 IN VIVO TRACING OF EXTRACELLULAR VESICLES IN ACUTE KIDNEY INJURY
Authors: Jiang Wei , Wang Bin , Liu Bicheng 
Published: June 2023

Abstract

Background and Aims
Extracellular vesicles, with their unique ability to migrate, target, and selectively internalize into specific cells, open up a new field of diagnosis, treatment, and drug delivery. However, a major disadvantage of translating exosome therapy into the clinic is that not enough is known about these endogenous nanovesicles, especially in vivo, and the tracing of extracellular vesicles in vivo can yield important information about their biological distribution, migration capacity, toxicity, biological role and mechanism of action. Therefore, to develop efficient, sensitive and biocompatible exosome markers. Recording and imaging techniques are much needed. Recent studies have developed different exosome labeling and imaging methods. However, due to the physiological location of the kidney and the distribution characteristics of extracellular vesicles in vivo, the specificity of live imaging of extracellular vesicles in the kidney is insufficient. Therefore, this study intended to construct a reliable and specific in vivo imaging method of extracellular vesicles in the kidney.

Method
Dual-mode imaging was used to trace extracellular vesicles in mice model of kidney disease, providing important technical support for tracing extracellular vesicles in vivo kidney and related drug delivery. Firstly, extracellular vesicles were separated for fluorescence staining, and the concentration variation rule of MPO, a disease-specific enzyme, was mastered in mouse kidney tissue. MPO, Luminol, DiI and DiD were used to construct a near-infrared imaging mode based on fluorescence resonance energy transfer to enhance fluorescence penetration, and targeted peptides were used to increase the aggregation of extracellular vesicles in kidney. This combines the sensitivity of fluorescence imaging with the specificity of kidney targeting peptides to reach deep tissues of extracellular vesicles.

Results
In the optical mode in vitro experiments, we found that exosomes could be co-stained by DIL and DID. In addition, the dye ratio of 3:7 could make exosomes exhibit the strongest excitation light. In the presence of luminol sodium, the fluorescence intensity of exosomes co-stained by DII and DID liphilic dyes was higher than that of exosomes with single dye. In the second mode of MRI, we found that our material can be efficiently adsorbed on exosomes by electron microscopy. Follow-up in vivo experiments are being further improved.

Conclusion
In this study, we constructed in vivo imaging of extracellular vesicles in the kidney based on optical imaging. Chemiluminescence generated by inflammatory specific expression of MPO and detection of MPO enzyme activity and specific detection substance luminol in the kidney disease model formed fluorescence resonance energy transfer in extracellular vesicles of double-stained cells. Preliminary efficacy evaluation showed that the fluorescence intensity of the double-dyed extracellular vesicles system in this model is greater than that of the single-dyed extracellular vesicles. This method can be applied to the in vivo tracer of extracellular vesicles in the kidney, providing technical support for the treatment of extracellular vesicles in kidney disease and the study of drug delivery.