Biomedical Engineering Reference
In-Depth Information
agents based on polyethyleneimine (PeI), polylysine, or other polyamine-based NP
have also been extensively studied as antisense delivery agents [120-122]. Because
of their high positive charge, they interact strongly with the cell membrane and
become endocytosed through one or more of many possible endocytotic mecha-
nisms, such as pinocytosis, macropinocytosis, clathrin-mediated, and caveolin-medi-
ated mechanisms [123, 124]. Both cationic lipid- and polymer-based systems show
toxicity, however, and will require further development before they can be used in
humans [120, 125]. Pretargeting and endocytosis of NP can also be achieved by
receptor-mediated endocytosis [126]. To this end, the folate and ferritin have been
widely used to deliver cargo into cancer cells [127, 128].
In addition to electrostatic binding, antisense agents can also be covalently
attached to the NP through noncleavable linkers, such as to gold NP through thiol-
gold bonds (Fig. 13.8c). Such gold-ODN NP are actively taken up by cells, presum-
ably by binding to serum proteins that are then endocytosed [129]. Alternatively,
antisense agents can be attached via other covalent reactions, such as amide bond
formation (Fig. 13.8d) or through an acid-labile or bioreductively cleavable linker
such as a disulfide bond (Fig. 13.8e). The specific linker used will depend on whether
or not the NP would hinder binding of the antisense probe to the target mRNA.
Having the antisense agent covalently bound to the NP runs the risk, however, that
much of it may remain trapped in endosomes along with the NP. This appeared
to be the case in a study with shell cross-linked NP linked to antisense PNAs via
an amide linkage that did not show bioactivity, whereas the same PNAs linked via a
bioreductively cleavable disulfide linker did [130]. confocal studies showed that
while the PNA was able to exit the endocytic compartments and enter the cytoplasm,
the NP was not. equally problematic with a noncleavable linker is that excess
unbound or partially bound NP may not be able to exit the cell leading to a high
background signal with an always -on probe. even with turn-on NP probes, the NP
could become occluded with target mRNA that could prevent the binding of other
target mRNAs. given these problems, it makes more sense to have an intracellularly
releasable antisense agent, unless one wants to use an attached NP to restrict the
probe from entering and accumulating in certain organelles. For example, tethering a
molecular beacon probe to a large protein complex was found to prevent unwanted
nonspecific accumulation of the probe in the nucleus [131]. In the case of binary
probes, it would make most sense to bind them to separate NP to prevent any inter-
action that might lead to a false signal.
The general problem with endocytosis as a mechanism for intracellular delivery is
that endocytosed NP generally become trapped in the endosomal/lysosomal pathway
and do not enter the cytoplasm where the mRNA targets are. Thus, the NP need to
incorporate additional functionality to enable the NP to escape the endosomes or at
least facilitating the exit of the probes. many amine-based NP appear to cause endo-
somal rupture by the pH sponge effect, which occurs as a result of proton pumps that
acidify the interior of endosomes to a pH of 4-5 [132-134]. NP that contain a high
concentration of amines have a number of amines that remain unprotonated at neutral
pH but can be protonated at lower pH (a proton sponge). As a consequence, the NP
function as a buffer that resists the lowering of the pH of the endosome and results
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