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.