methods currently developed have some limitations. Pros and cons of each method
is summarized in Table 8.1.
8.1.2 Fluorescence Labeling
Direct fluorescent-dye labeling have been most widely used for the labeling of EVs.
Fluorescence labeling enables whole body imaging of small animals using highly
sensitive optical cameras and fluorescence microscopic imaging. Lipophilic dyes
such as PKH, DiI and DiR can be used for fluorescence emitting EVs [11–16]. This
simple labeling method can visualize cellular-level interactions. For example,
live-cell imaging showed internalizing EVs through endocytosis pathway and
recycling of EVs [15]. However, fluorescence dye imaging is limited to exogenous
EVs. Furthermore, it is unable to acquire deep organ imaging. In terms of in vivo
imaging, among lipophilic dyes, near-infrared (NIR) dyes are more ideal for in vivo
imaging than general fluorescence dye as NIR could provide higher signal-to-noise
ratio and relatively higher penetration depth. For example, a report used lipophilic
NIR dye, IRDye800, for whole body biodistribution study [17]. Nonetheless, the
method has inherent limitation against direct clinical translation. In addition,
recently, issues with regard to tracking biodistribution using lipophilic dyes in
preclinical studies have been raised. EVs can be metabolized and degraded in
in vivo, however, dyes persist in tissues and emit signals, which results in misleading results of biodistribution. Lipophilic dyes could be integrated with intact
lipid bilayers as well as degraded lipids and other lipophilic compounds during
circulation [9]. Furthermore, lipophilic dyes may cause aggregation of EVs and
hinder accurate imaging and trafficking of EVs [18].
As aforementioned, reporter system can be used for fluorescence imaging of
EVs. Protein markers of EVs, such as CD63, were used to design reporter conjugated to fluorescent proteins [19, 20]. This labeling strategy allows to generate a
cell line that continuously produce EVs containing the reporter proteins. In spite of
the stability, the signal intensity depends on the amount of protein expression of
EVs, which could be heterogeneous in population of EVs. Subpopulation of EVs
without specific reporter protein cannot be monitored by this specific
protein-targeted reporter system [21]. Another reporter method based on palmitoylation signal provided membrane of EVs expressing fluorescence signals [9].
While lipophilic dyes can be nonspecifically bound to lipid entities as well as EVs,
the reporter system has specific and selective imaging of EVs. This property allows
for live-cell imaging of EVs from specific cell types which showed exchanging EVs
between different cell populations [22]. However, they need genetically engineered
cells which limit in clinical application as well as flexible labeling for various types
of EVs. Whole body imaging instead of microscopic cellular level imaging using
this method is difficult due to relatively low yield of fluorescence-labeled EVs from
reporter system.
8 Endogenous Radionanomedicine: Biodistribution and Imaging
155
is summarized in Table 8.1.
8.1.2 Fluorescence Labeling
Direct fluorescent-dye labeling have been most widely used for the labeling of EVs.
Fluorescence labeling enables whole body imaging of small animals using highly
sensitive optical cameras and fluorescence microscopic imaging. Lipophilic dyes
such as PKH, DiI and DiR can be used for fluorescence emitting EVs [11–16]. This
simple labeling method can visualize cellular-level interactions. For example,
live-cell imaging showed internalizing EVs through endocytosis pathway and
recycling of EVs [15]. However, fluorescence dye imaging is limited to exogenous
EVs. Furthermore, it is unable to acquire deep organ imaging. In terms of in vivo
imaging, among lipophilic dyes, near-infrared (NIR) dyes are more ideal for in vivo
imaging than general fluorescence dye as NIR could provide higher signal-to-noise
ratio and relatively higher penetration depth. For example, a report used lipophilic
NIR dye, IRDye800, for whole body biodistribution study [17]. Nonetheless, the
method has inherent limitation against direct clinical translation. In addition,
recently, issues with regard to tracking biodistribution using lipophilic dyes in
preclinical studies have been raised. EVs can be metabolized and degraded in
in vivo, however, dyes persist in tissues and emit signals, which results in misleading results of biodistribution. Lipophilic dyes could be integrated with intact
lipid bilayers as well as degraded lipids and other lipophilic compounds during
circulation [9]. Furthermore, lipophilic dyes may cause aggregation of EVs and
hinder accurate imaging and trafficking of EVs [18].
As aforementioned, reporter system can be used for fluorescence imaging of
EVs. Protein markers of EVs, such as CD63, were used to design reporter conjugated to fluorescent proteins [19, 20]. This labeling strategy allows to generate a
cell line that continuously produce EVs containing the reporter proteins. In spite of
the stability, the signal intensity depends on the amount of protein expression of
EVs, which could be heterogeneous in population of EVs. Subpopulation of EVs
without specific reporter protein cannot be monitored by this specific
protein-targeted reporter system [21]. Another reporter method based on palmitoylation signal provided membrane of EVs expressing fluorescence signals [9].
While lipophilic dyes can be nonspecifically bound to lipid entities as well as EVs,
the reporter system has specific and selective imaging of EVs. This property allows
for live-cell imaging of EVs from specific cell types which showed exchanging EVs
between different cell populations [22]. However, they need genetically engineered
cells which limit in clinical application as well as flexible labeling for various types
of EVs. Whole body imaging instead of microscopic cellular level imaging using
this method is difficult due to relatively low yield of fluorescence-labeled EVs from
reporter system.
8 Endogenous Radionanomedicine: Biodistribution and Imaging
155