Biomedical Engineering Reference
In-Depth Information
the cell surface, Tf conjugates commonly are efficiently delivered to various intra-
cellular locations. For these reasons, Tf (or TfR binding antibodies and peptides)
has been used as targeting ligand for drug, protein, and gene delivery [
95
], includ-
ing the delivery of pDNA [
68
,
88
,
96
-
105
] and siRNA [
106
-
108
].
In most cases Tf has been incorporated into polyplexes by direct conjugation to a
DNA binding molecule. Ethidium homodimer [
99
] and many polycations, includ-
ing PLL and protamine [
96
-
98
], PEI [
68
,
100
], polypropylenimine (PPI) dendrimer
[
104
], or PAMAM dendrimer [
103
], have been used for this purpose. Coupling has
been performed by modification of Tf lysine amines with bifunctional linkers [
97
]
or via periodate-oxidized carbohydrates [
99
,
102
] of Tf. In some cases, a bridging
PEG linker has been applied [
68
,
103
]. Tf has been attached to polyplexes also in
a more indirect way. Activated Tf has been added to polyplexes after polyplex
formation [
109
], Tf has been conjugated to polyplex surface-shielding copolymers
[
82
] or applied as a PEG-adamantane conjugate for noncovalent attachment to
cationic
b
-cyclodextrin-based polymers [
101
,
110
].
Epidermal growth factor receptor (EGFR) is another receptor that is upregulated
in several cancers, such as glioblastoma, lung or colorectal cancer. Various EGFR
binding molecules have been explored as targeting ligands in polyplexes, including
recombinant EGF protein [
111
-
114
], EGFR binding antibodies [
115
], and peptides
[
116
,
117
]. For EGF/PEG/LPEI polyplexes, the cellular uptake process was
evaluated in detail by real-time single particle fluorescence microscopy. HUH7
hepatoma cells expressing high EGFR levels were used in recombinant version,
with either enhanced green fluorescent protein (eGFP)-tagged actin (for visualiza-
tion of the actin cytoskeleton network) or eGFP-tagged tubulin (for visualization of
the microtubules). In the initial phase, both targeted and untargeted polyplexes dock
to the cell surface and slowly diffuse along the cell surface simultaneously with the
actin cytoskeleton, presumably via transmembrane contacts (mediated by trans-
membrane adaptor molecules). The presence of the receptor ligand EGF strongly
accelerated the intracellular uptake of polyplexes. After 5 min, 50% of the EGF-
targeted polyplexes were internalized, whereas less than 10% of untargeted poly-
plexes were internalized at this time point [
114
]. Within the cells, polyplexes were
found within endosomes, which migrate along the microtubule cytoskeleton.
Numerous other ligands have been evaluated for polymer-based tumor cell
targeting [
118
-
124
]. For example, bombesin, a peptide that binds bombesin receptors
expressed in tumors such as small cell lung carcinoma and gastric cancer, was used as
ligand for siRNA polyplex delivery [
122
]. The folate receptor is upregulated
in various cancers. Thus, the natural low molecular weight targeting molecule folic
acid was applied for delivery of polymer-based pDNA [
123
] and siRNA, with siRNA
covalently bound to folate via a short linker [
124
].
The neoangiogenic tumor vasculature overexpresses certain integrins and other
surface markers, which can also be used for targeting of polyplexes. The RGD
peptide motif has been successfully applied for integrin-targeted pDNA [
125
-
128
]
and siRNA [
129
,
130
] delivery. In many cases, the PEG motif-containing peptide
was attached to the polycation via a PEG spacer. For RGD-PEG-PEI/pDNA
polyplexes, an optimum grafting with RGD-PEG was required because transfection
