The Application of Plant Viral Nanoparticles in Tissue-Specific Imaging - Nanotechnology for Biomedical Imaging and Diagnostics - page 402

is the installation of nucleic acid aptamers on the viral capsid. Using the systematic
evolution of ligands by exponential enrichment (SeLeX) process, aptamer sequences
can be developed that can bind to any chosen target with high specificity. Cell-
specific imaging was demonstrated using MS2 mutants with p -aminophenylalanine
( p aF) and cysteine amino acids inserted in their coat proteins on their exterior and
interior surface, respectively. p aF allows for an efficient oxidative coupling strategy
to be employed for aptamer attachment onto the capsid surface, while cysteine per-
mits interior labeling through maleimide chemistry. As a preliminary proof of con-
cept, a 41-nucleotide sequence that targets tyrosine kinase receptors on Jurkat T cells
was substituted with phenylenediamine and coupled with the aniline groups of p aF
in the presence of sodium periodate (Naio 4 ), while a fluorescent dye was installed
within the capsid. The particles were observed using confocal microscopy, and
receptor-mediated internalization was confirmed, demonstrating that the installation
of aptamers on VNps is a promising method for the localization of specific cellular
markers and cell imaging [88].
other recent studies focused on engineering VNp formulations targeted to bio-
markers that are preferentially expressed in tumors. The folate receptor (Fr) is one
such marker that is upregulated on cancer cells as a result of greater demand for folic
acid (FA) during proliferation. FA is a vitamin essential for DNA replication, cell
division, and growth, and FA uptake is mediated by binding to Fr [89]. Conjugation
of FA to VNps was first investigated using CpMV and evaluated with KB and heLa
cancer cell lines. KB is a nasopharyngeal cancer cell line with high expression of Fr,
while heLa is a cervical cancer cell line with reduced Fr expression. Cells were
incubated with the CpMV particles at 4°C to minimize endocytosis and monitored
by flow cytometry for particle binding after permeabilization and treatment with
anti-CpMV primary and fluorescent secondary antibodies. Direct conjugation of FA
to surface lysines did not result in any improvement in cellular binding, which may
be due to FA being inaccessible so close to the capsid or possibly native CpMV-cell
interactions. presentation of FA using a peG linker resolved the difficulty and
resulted in up to 94% binding to KB cells and a modest 22% binding to heLa cells,
while control formulations with just peG showed negligible binding. A competition-
binding assay using free FA demonstrated that binding of CpMV-FA was disrupted
in the presence of free FA, indicating lack of a multivalent avidity effect. regardless,
conjugation of FA showed significant improvement in cell binding compared to FA
alone, thus making CpMV-peG-FA an attractive targeted design for cell-specific
imaging [90].
Another possibility for a targeting ligand that could be displayed on VNps is
transferrin (Tfn), an 80kDa iron-binding glycoprotein. Tfn receptors (Tfnrs) are
expressed at high levels on a variety of tumor cells and serve to transport Tfn loaded
with iron into the cell through clathrin-mediated endocytosis [91]. Functionalization
of Tfn for conjugation to VNps can be accomplished through oxidation of its sialic
acid residues. This method was used to make bacteriophage hK97 labeled with Tfn,
and uptake of hK97-Tfn in tumor cells was compared with that of native hK97 using
flow cytometry and anti-hK97 antibody staining. The nontargeted particles did not
interact with the cells, possibly as a result of its strong negative surface charge. on