The Application of Plant Viral Nanoparticles in Tissue-Specific Imaging - Nanotechnology for Biomedical Imaging and Diagnostics - page 404

intravital imaging was also demonstrated using a targeted formulation incorpo-
rating the bombesin peptide. Bombesin targets the gastrin-releasing peptide receptor
(Grpr), which is one of the most overexpressed receptors in human cancers,
including prostate cancer [96]. CpMV nanoparticles functionalized with either peG
or peG-bombesin and a Nir dye, Alexa Fluor 647, were utilized to analyze targeting
specificity to pC-3 prostate cancer cells. In vitro studies using fluorescence confocal
microscopy imaging showed that CpMV-peG did not bind to the pC-3 cells at a
noticeable level, while there was significant binding of CpMV-peG-bombesin.
A competition-binding assay using a 10-fold excess of free bombesin significantly
decreased the uptake of CpMV-peG-bombesin, demonstrating that internalization
is due to bombesin-Grpr interactions. Targeting specificity was also evaluated
using flow cytometry, and as expected, CpMV-peG-bombesin had significantly
higher uptake compared to CpMV-peG. Nonspecific uptake of CpMV-peG was
somewhat elevated, most likely the result of insufficient peG coverage to completely
shield the CpMV-vimentin interaction. Targeting was also examined in vivo using
the CAM model implanted with GFp-expressing pC-3 cells and intravital imaging.
After intravenous injection, CpMV-peG was quickly cleared from the tumor, while
CpMV-peG-bombesin accumulated over a period of 6 h, with low background in
the surrounding vasculature (Fig. 14.5). These observations indicate that CpMV-
peG-bombesin nanoparticles would be valuable for molecular imaging of Grpr-
overexpressing tumors [69].
Targeted VNp formulations for molecular imaging have recently been described,
and it is clear that there are many targeting ligands that can be incorporated for
customized detection of a wide range of diseases. VNps are fairly simple to modify,
with several robust bio-orthogonal chemistries established for functionalization.
As imaging techniques continue to improve, new fluorescent dyes developed, and
targeting ligands discovered, targeted VNps for in vivo imaging and diagnostics
will continue to progress.

14.6 Vnps As mri conTrAsT AgenTs

Mri is used regularly as a noninvasive diagnostic tool, with the advantage of not
requiring the use of ionizing radiation. Mri works by aligning protons of water mol-
ecules in a magnetic field and then exciting the protons with a pulse of radio-fre-
quency (rF) waves applied perpendicular to the field. After the rF pulse, the protons
“relax” and realign themselves with the magnetic field. The energy that is released
during this relaxation process is detected and used to generate the Mri image. The
return of the protons to equilibrium is typically characterized by two relaxation times,
T 1 and T 2 , where T 1 is the time constant describing the realignment of the protons with
the magnetic field and T 2 is the time constant related to the loss of phase coherence
between the protons that was instigated by the rF pulse (reviewed in ref. [97]).
Contrast agents are typically used to shorten the relaxation time of nearby water
protons in locations of disease, appearing bright T 1 -weighted images and dark T 2 -
weighted images. in this manner, the difference in the area of disease is enhanced and