The Application of Plant Viral Nanoparticles in Tissue-Specific Imaging - Nanotechnology for Biomedical Imaging and Diagnostics - page 403

the other hand, hK97-Tfn showed high levels of internalization in both cell lines
tested, heLa and hT-29. internalization was shown to be Tfnr-mediated using a
competition-binding assay with free Tfn. receptor-mediated endocytosis was further
confirmed with visualization by confocal microscopy of hK97 dual-labeled with Tfn
and fluorescent dye oregon Green 488 (o488). intracellular colocalization with
LAMp-1 was observed, indicating that hK97-Tfn-o488 is transported into the
endolysosomes following endocytosis [92]. This finding is in agreement with another
investigation using a different Tfn-labeled bacteriophage, Qβ. Qβ-Tfn conjugates
and native Qβ were labeled with a fluorescent dye, Alexa Fluor 568, and the particles
were incubated with African green monkey kidney epithelial cells (BSC1) that
express eGFp-labeled clathrin light chains (Clc) to study clathrin-mediated endocy-
tosis. incubation at 4°C followed by 37°C was used to determine binding of the par-
ticles to the cells and internalization, respectively. No binding or internalization was
found for Qβ, while significant binding and uptake were found for Qβ-Tfn. The Qβ-
Tfn colocalized with the clathrin-coated pits, and there was no binding or uptake
observed when excess free Tfn was present, indicating that the internalization
occurred through a Tfnr-mediated endocytosis. This was further confirmed using
total internal reflection fluorescence (TirF) live cell microscopy, where the bottom
surface of the cell membrane was imaged and the internalization of Qβ-Tfn through
clathrin-coated pits was observed. Different ligand densities of Tfn were compared,
and although the rate of uptake increased with more Tfn molecules loaded, there was
not a significant increase in affinity on a per-Tfn basis. Nevertheless, on a per-parti-
cle basis, the Qβ-Tfn conjugates have greater affinities than free Tfn, which could be
advantageous for targeted imaging of cells and delivery of therapeutics [93].
Although targeted platforms have been demonstrated mainly in vitro , they have
also recently been validated in vivo . F56 is a synthetic peptide discovered through
phage display that binds with high affinity to vascular endothelial growth factor
receptor-1 (VeGFr-1) and as a result significantly hinders tumor angiogenesis
and metastasis [94]. Multifunctional particles were made by attaching fluorescent
peG-fluorescein and F56-fluorescein to CpMV. Fibroblasts that do not express
VeGFr-1 and human endothelial cells with high VeGFr-1 expression were treated
with CpMV-peG and CpMV-F56 conjugates and evaluated by confocal fluorescent
imaging. The data demonstrated that the F56 peptide successfully targets the CpMV
particles to endothelial cells, with no nonspecific binding to the fibroblast cells and
no binding observed for CpMV-peG. Additional studies ex vivo and in vivo were
then performed with hT-29 colon cancer cells, which express high levels of VeGFr-
1. Confocal microscopy was used to investigate binding of the particles to hT-29
tumor sections ex vivo , and there was significant binding to the tumor sections with
F56 conjugates but not with peG. Similar observations were found in vivo after intra-
venous injection of the particles in nude mice bearing hT-29 tumor xenografts. The
tumors were sectioned and confocal microscopy was performed. No cellular uptake
was found for CpMV-peG, but accumulation throughout the tumor was observed for
CpMV-F56 particles. overall, confocal microscopy established that targeting with
F56 is effective in vitro , ex vivo , and in vivo , although alternate dyes with better tissue
penetration would be required for imaging in vivo [95].