Background

Exosomes are natural nanovesicles (30-150 nm in diameter) of endosomal origin, secreted by a variety of cells including mesenchymal stem cells (MSCs) (Yang et al., 2018). Exosomes carry proteins, lipids and genetic materials of parent cells, and are capable of inducing a multitude of biological effects to adjacent or distal cells and tissues (Valadi et al., 2007; van Niel et al., 2018). Due to their innate advantages, such as low immunogenicity, potential of crossing blood brain barrier, and amenability to the loading of exogenous cargos, exosomes have emerged as an alternative to cell-based therapeutics in the treatment of cancer, neurodegenerative and many other diseases (Alvarez-Erviti et al., 2011; Kamerkar et al., 2017; Guo et al., 2019; Yong et al., 2019). While exosomes have gained increasing interest as diagnostic and therapeutic vehicles, the knowledge of their in vivo behavior is limited, which restricts the clinical translation of exosome therapy. Thus, advanced imaging approaches for in vivo tracking of exosomes are highly anticipated.

To elucidate the in vivo behavior of exosomes, several strategies have been developed. Among them, optical imaging represents the most commonly used exosome tracking method. Up until now, organic dyes, immunofluorescent reporters, genetically encoded fluorescent proteins, or fluorescent nanomaterials have been used to label exosomes (Pegtel et al., 2010; Alvarez-Erviti et al., 2011; Suetsugu et al., 2013; Lai et al., 2015; Jiang et al., 2018; Shen et al., 2018; Betzer et al., 2019). Optical imaging allows hgh-throughput efficiency, facilitating the observation of the interplay between exosomes and recipient cells. However, this technique has several drawbacks, such as artifacts due to lipophilic dye aggregation (Grange et al., 2014) or presence of autofluorescence background (Faruqu et al., 2019), as well as limited tissue penetration depth (Shen et al., 2018). Magnetic resonance imaging (MRI) constitutes another promising tracking methodology, featured by high spatial resolution with anatomical details and radiation-free operation (Busato et al., 2017). Exosomes can be loaded with superparamagnetic iron oxide nanoparticles (SPIONs), and tracked in animal models using MRI (Hu et al., 2015; Busato et al., 2016; Jia et al., 2018). MRI, however, has inherent disadvantages, including susceptibility to movement artifacts, long image acquisition, and high cost. Computed tomography (CT) is a powerful, and widely used imaging modality which offers high spatial and temporal resolution. Recently, Betzer et al established a protocol for labeling exosomes with gold nanoparticles (GNPs) for tracking exosome migration in vivo using CT (Betzer et al., 2018). This technology has been successfully utilized to track the biodistribution of intranasally delivered exosomes derived from mesenchymal stem cell (MSC), in rodents with focal brain ischemia (Betzer et al., 2017), various brain pathologies (namely, stroke, Parkinson’s disease, Alzheimer’s disease, or autism) (Perets et al., 2019), or spinal cord injury (SCI) (Guo et al., 2019). Guo et al combined optical imaging and CT neuroimaging modalities, to examine the biodistribution of intranasally administered MSC-derived exosomes in rats with complete SCI (Guo et al., 2019). Coupled with quantitative methodologies, i.e., inductively coupled plasma (ICP), flame atomic absorption spectroscopy (FAAS), and analysis of immunofluorescently stained tissues, a more comprehensive exosome tracking was demonstrated, unraveling the interactions between exosomes and different cell populations in the spinal cord, and the localization of exosomes in several major organs, in the context of SCI.

Multimodal imaging modalities are more anticipated than single imaging model with regard to elucidating the behavior of exosomes in vivo through complementary advantages. The protocol presented here can be expanded to investigate how exosomes behave in other diseased models.