Intracellular barriers
The cellular uptake is the first intracellular barrier that the delivery
systems encounter (Figure 3). Nanoparticle-based delivery systems should
interact with components of the outer surface of cells for
internalization into them. To date, five recognized mechanisms have been
proposed for the uptake of nanoparticles based on the proteins involved
in the endocytosis process; micropinocytosis, phagocytosis,
caveolin-mediated, clathrin-mediated, and clathrin/caveolin-independent
endocytosis (Sahay, Alakhova, & Kabanov, 2010; Q. Sun et al., 2019).
The central parameters determining the endocytic pathway are morphology,
surface chemistry, and size. These factors can affect both cellular
uptake efficiency and the endocytic route (J. Zhao & Stenzel, 2018). To
improve the EPR effect, 30-100 nm-sized multimolecular
nanoparticle-based delivery systems have been typically designed,
including polymeric micelles and lipid nanoparticles (LNPs). Moreover,
size of rigid nanoparticles affects their endocytosis; 20-50 nm-sized
bare gold nanoparticles have the highest cellular uptake in pancreatic
cancer cells (H. Gao, Shi, & Freund, 2005). Surface chemistries of
nanoparticle-based delivery systems are much more important for their
endocytosis, in comparison with shape and size. Cationic
nanoparticle-based drug delivery systems have a high affinity for
anionic proteoglycans presented on the membrane of various cells,
leading to more effective adsorptive endocytosis than anionic and
neutral nanoparticle-based drug delivery systems. It is noteworthy that
heparan sulfate proteoglycans, consisting of transmembrane proteins
known as syndecans, are regarded as the main binding sites for
positively charged
nanoparticle-based drug delivery
systems (Zhi et al., 2018). But the excess positive charge can lead to
some serious problems such as rapid opsonization and clearance,
toxicity, and increased immunological reactions. To reduce aggregation
of particles and consequently overcome the aforementioned problems,
PEGylation of nanoparticle-based drug delivery systems is a common
approach (Suk, Xu, Kim, Hanes, & Ensign, 2016). Nevertheless, PEGylated
delivery systems still have some limitations, including immunogenicity
and low cellular uptake and endosomal escape (Lai & Wong, 2018).
To address these limitations, a vast amount of research devoted to the
design of nanocarriers with cell recognition moieties (ligand-mediated
targeting strategies) to improve their cellular uptake through
receptor-mediated endocytosis. On the other hand, these nanoparticles
can be designed to lose their PEG-shell at cell surfaces or in the
acidic endosomes of cells to increase their release into the cytoplasm
(Öztürk-Atar, Eroğlu, & Çalış, 2018).
Entrapment of nanoparticle-based siRNA delivery systems into the
vesicles, including early endosome (pH 6.5), late endosome (pH 6.0), and
lysosome (pH 4.5–5.0), often leads to their breakdown due to their
susceptibility to acidic environment and the involved enzymes (S. A.
Smith, Selby, Johnston, & Such, 2019). Hence, the design of delivery
systems should facilitate its entry from vesicles into the cytoplasm
(Figure 3). Numerous works have established that polycations comprising
amines with low pKa and their polyion complexes (PICs) with siRNAs can
induce endosomal escape via two mechanisms. First, some of these low pKa
amines have a role in electrostatic interaction with anionic siRNAs
whereas the protonation of unbound amine groups can take place in acidic
pH of the endosomal compartments (B. S. Kim et al., 2019). Through this
process, influx of protons along with chloride ions is induced. As a
result, the osmotic pressure increases in endo/lysosomal vesicles
inducing the endosomal disruption defined as the “proton sponge
effect”. Another proposed mode of action is that these highly charged
polycations can directly destabilize endosomal membrane (Koide et al.,
2019). As formerly mentioned, polycations can interact with the
negatively charged cytomembrane and disrupt membrane integrity.
Polycations, which bear low pKa amines can considerably increase their
cation density via amine protonation in endo/lysosomal vesicles and thus
destabilize the membrane of endo/lysosome.
Figure 3. Schematic
illustration of intracellular barriers in pancreatic cancer siRNA
delivery.