Biomedical Engineering Reference
In-Depth Information
localization [63, 64]. Furthermore, for VNps displaying targeted ligands, dye labeling
and flow cytometry can be used together to make high-throughput measurements of
specificity of uptake [37].While chemical conjugation to the protein coat is the most
common technique for attaching dyes, several other methods can also be employed.
encapsulation as described previously is one such method for forming optical
constructs for intracellular imaging. it was found that dyes could be infused into
rCNMV through gating by altering the ph and divalent ion concentrations to open
or close the pores. The efficiency was charge dependent, with high loading of posi-
tively charged and neutral molecules but negligible loading of negatively charged
dyes, likely due to their interaction with the rNA within the capsid interior [26].
encapsulation through a reassembly process has also been demonstrated. Different
types of molecular coatings, such as lipid micelles, streptavidin-biotin-DNA, dihy-
drolipoic acid (DhLA), and carboxylic peG thiol, were investigated for encapsu-
lating CdSe/ZnS quantum dots (QDs) in BMV. QDs functionalized with peG were
found to have the best reassembly efficiency and photostability [65]. Labeling BMV
with fluorescent dyes through reassembly has also been demonstrated using an
FDA-approved Nir fluorophore indocyanine green (iCG), which has an intrinsic
negative charge to trigger its encapsulation without requiring a coating [66].
Noncovalent heterodimeric coiled-coil protein interactions can also be applied to
couple fluorescent proteins with the capsid. By modifying the coiled-coil motif with
glutamic acid (e-coil) or lysine (K-coil), heterodimerization from the association of
the e-coil with the K-coil can be achieved. This was demonstrated using the CCMV
capsid, introducing a K-coil at the N-terminus of its coat protein and an e-coil at the
C-terminus of enhanced green fluorescent protein (eGFp) through genetic engi-
neering. Complexes of the modified protein subunits and eGFp were formed
in vitro
.
The eGFp-capsid protein complexes were then mixed with wild-type capsid proteins
at various stoichiometric ratios to assemble CCMV capsids with good control over
the amount of protein encapsulated, with up to 15 eGFp per particle obtained [67].
preliminary dye-labeling studies such as these are crucial to the development of
VNp molecular probes. Fluorescent VNps modified with additional functionalities
have been applied for visualizing biodistribution of the particles as well as for detect-
ing and monitoring disease [15].
14.3 inTerAcTion of cpmV wiTh cells And iTs ApplicATion
for inTrAViTAl imAging
CpMV nanoparticles have been studied extensively
in vivo
and applied for potential
applications in biomedicine [18, 53, 68-70]. CpMV has been shown to recognize
a variety of mammalian cells, such as murine fibroblast, pC-3, heLa, and hT-29
cells, through their interaction with vimentin, a type iii intermediate filament that
is responsible for modulating the architecture and dynamics of cells [54, 71-73].
Vimentin is a cytoskeletal protein that is important for various cellular functions,
including wound healing, cell adhesion, cell migration, proliferation, protein syn-
thesis, gene expression, and signal transduction [74, 75].
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