ENGINEERING OF CELL-PENETRATING PEPTIDE-CONJUGATED INTRACELLULAR DELIVERY SYSTEMS - Biologically-Responsive Hybrid Biomaterials - page 209

oxide particles (CLIO) (41nm). The peptide was attached to CLIO amine,
activated with succinimidyl-iodoacetate. These particles enabled internalization
into cell lines tested (mouse lymphocytes, human natural killer cells and HeLa
cells) over 100-fold more efficiently when compared with TAT-free controls.
Later in another study, T cells were magnetically labeled without affecting their
normal responses to stimulation [86]. Upon the intravenous injection, the homing
of T cells to the spleen could be detected by MRI and their biodistribution profile
could be followed by MRI in vivo . Other studies observed the uptake of TATp-
labeled iron oxide particles by T cells, B cells, and macrophages over 72 h and
found that the uptake was quick with no loss in the cell viability [87]. However
the TATp-iron oxide conjugates were accumulated primarily in the cytoplasm in
contrast to nuclear localization observed by other authors. In general, the labeling
with TATp-CLIO did not induce toxicity and did not alter the differentiation or
proliferation pattern of CD34+ cells. The TATp-CLIO-labeled CD34+ cells and
control cells were subsequently injected intravenously into the immunodeficient
mice [88]. Around 4% of the injected dose of the cells migrated to the bone
marrow (per gram of the tissue), and the labeled and control cells showed similar
biodistribution profile. Nonetheless, it was possible to visualize the labeled cells
by MRI within mouse bone marrow at the single-cell level. Also, such
magnetically labeled cells could be recovered from the bone marrow after in vivo
homing using magnetic separation columns. Work by Zhao et al. suggested that
the cellular uptake of the TATp-CLIO increased nonlinearly upon increasing
TATp-to-CLIO ratios, giving a 100- fold increase with 15 TATp per CLIO
particle thus enhancing the sensitivity of imaging by MRI [89]. Work carried by
Lewin et al. showed that TATp-functionalized superparamagnetic nanoparticles
were able to track labeled cells in vivo using MRI and also could recover the
cells using magnetic-separation column [55]. When the surface of CLIO was
modified with D-polyarginyl peptide or TATp, the nanoparticles could traverse
through the cell monolayers. Such nanoparticles can be used, for example, for
labeling of immune cells for in vivo imaging purposes [90].
The same approach was applied to prepare octaarginine-coated liposomes to
target airway cells by inhalation [91]. In another study, shell cross-linked (SCK)
nanoparticles were prepared by the micellization of amphiphilic block
copolymers of poly(epsilon-caprolactone-beta-acrylic acid) and conjugated to
TATp that was independently built on a solid support, resulting in TATp-
modified nanocage conjugate. Such a conjugate when analyzed by confocal
microscopy with CHO and HeLa cells, showed binding and transduction inside
the cells [92]. The authors then characterized the SCK nanoparticles for the
optimum number of TATp per particle required to enhance the transduction