**7. Strategies for endosomal escape of cargoes**

*Role of Novel Drug Delivery Vehicles in Nanobiomedicine*

Above mentioned limitations, *viz.*, altered 3D conformation, undesired aptamer selection against the protein-tag or matrix can be overcome by using cell-SELEX methodology. In cell-SELEX aptamer selection is performed on live cells *in vitro*, hence, target surface proteins are present in their native conformation. Unlike protein-based SELEX, cell-SELEX does not need any prior information about the native conformation or biological function of the target protein. This method relies on the difference in the expression pattern of cell surface receptors between the diseased target cells (e.g., cancer cells) and the non-diseased cells (e.g., healthy cells). The aptamer selection is first performed on a negative control, i.e., healthy cells. From here, the unbound aptamers are collected and added to the test cell line, i.e., diseased cells. Only the unbound aptamers are collected from negative control cells because they are uncommon among the target and control cells, and it possibly has a set of aptamers that bind the upregulated cell surface receptors that are present only on target diseased cells. Using the above-mentioned methodology, cell-SELEX has been used to identify novel tumor biomarkers [65]. In our experience, major challenge of cell-SELEX is the optimization of aptamer enrichment by PCR as compared to protein-based SELEX. This is due to the inhibitors or other cellular contaminants (e.g., DNA or RNA) that come in the aptamer pool which inhibit the polymerasebased PCR reaction for enriching aptamer pool during each round of selection. Also, lack of prior knowledge about the identity and expression level of the biomarker may lead to aptamer selection against unrelated/unwanted off-target surface molecules that are co-expressed on target cells; hence, more rounds of counter selection with

the control cell line is required to improve the selectivity of aptamers.

An ideal aptamer candidate for internalization purpose should bind to a cell surface receptor that is present in high quantity, and it should have a high rate of endocytosis upon binding of a ligand. However, aptamers selected by cell-SELEX may not fall in this category always. Hence, researchers have developed novel strategies to facilitate enrichment of cell internalizing aptamers by alteration of the traditional cell-SELEX protocol. For this purpose, Thiel et al. developed a methodology called cell-internalization SELEX. Here, target cells were incubated with an RNA library followed by high-salt wash [66]. This method eliminates the non-internalizing surface bound aptamers or those that internalize at a slower rate. Cell internalizing aptamer against Human epidermal growth factor receptor (HER2), a transmembrane protein overexpressed in breast cancer cells was selected using this method [66]. Later studies reported that a high-salt wash is insufficient to remove the non-internalizing surface bound aptamers. Hence, Levy et al. (2012) used a cocktail of RNAses to digest the cell surface bound non-internalizing aptamers [67, 68]. Using this strategy, this group selected cell internalizing 2′-fluoropyrimidine RNA aptamer called c2 against human

Proteinase K is a broad-spectrum serine protease that cleaves peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha-amino groups. Proteinase K was used as an alternate approach to digest protein domains present on the cell surface after incubation with aptamer library. Thus, digestion of protein domains would render loss of cognate epitopes needed for aptamer binding. After proteinase K treatment and washing of the cells, the aptamers non-specifically bound to the cell surface proteins would be removed, and only those which have internalized the cells can be obtained, as desired. Proteinase K treatment can be used for DNA and RNA aptamers both, unlike RNase treatment used by Levy et al. [68] which is applicable only for RNA aptamers. Iaboni et al. used this method to select cell-internalizing aptamers against insulin receptors on

**6.2 Cell SELEX**

**10**

transferrin receptor (hTfR).

human glioma cells [69].

The therapeutic efficiency of a cell-internalizing platform depends on its ability to escape from endosomes and deliver the therapeutic cargo to the cytoplasm. The efficiency of endosomal escape, which is 0.01% for traditional delivery systems, should be increased significantly to achieve higher therapeutic efficacy [70]. Two types of strategies are commonly employed to facilitate endosomal escape. First method uses endosomal destabilizing agents that alter the permeability of endosomal membranes, e.g., chloroquine. However, the limitation of this method is that these endosomolytic agents may reach lysosomes, thus, lysing them and lead to cytotoxicity due to the release of lysosomal lytic enzymes. Second method, which is found to be non-toxic in several studies, uses engineered viral or bacterial cell penetrating peptides (CPPs) (e.g., truncated HIV-1 TAT peptide or Aurein1,2) [71–73]. However, CPPs can increase the escape of cargoes from endosomes to the cytoplasm by only 5- to 8-fold, which is not enough to generate an effective platform for clinical applications [70]. Below we will discuss the strategies that could be used with aptamers to enhance their endosomal escape properties or that of the attached cargo.

Any targeted delivery platform for internalization of a cargo to cells is endocytosed by receptor-mediated endocytosis (RME) to a large extent [74], and aptamers fall in this category. The endocytosed cargo first reaches early endosomes, from where it is either sent back to cell surface (recycling endosomes) or sent to late endosomes, and finally lysosomes. The cargo may be degraded by hydrolytic enzymes in lysosomes. Therefore, if a cargo enters an endosome that is destined for lysosomes, it has a short period of time within which it has to escape to the cytosol to prevent its degradation. Different strategies have been developed that can be used in conjugation with aptamers, which may help in their endosomal escape. Endosomal sorting complex required for transport-I (ESCRT-I) is a protein complex that plays an important role in maturation and trafficking of endosomal cargoes. Wagenaar et al. silenced two components of ESCRT-1 (i.e., TSG101 and VPS28) to induce endosomal escape of delivered anti-miRNA *in vitro* and *in vivo*, both, and resulted in tumor regression [75].

Nieman-Pick type C1 (NPC1) is a late endosomal/lysosomal membrane protein required for extracellular recycling of endosomal contents. A small molecule inhibitor of NPC1 protein, termed NP3.47, was earlier used to delay maturation of non-recycling endosomes [76]. The Study found that NP3.47 caused 3-fold higher accumulation of lipid nanoparticle (LNP) formulations of siRNA, i.e., LNP-siRNA as compared to the controls in late endosomes/lysosomes in a variety of cell lines causing 4-fold higher gene silencing. Authors proposed that trapping LNP-siRNA in late endosomes extended the window of time for a more efficient escape into the cytosol to facilitate siRNA induced gene silencing [76].

pH-dependent cargo release is also an effective way for endosomal escape. Anti-PSMA aptamer-siRNA chimera was conjugated to a pH-dependent polyhistidine domain *via* dsRNA binding domain (dsRBD). In the acidic compartment of a mature endosome (pH ≤ 6), Histidine becomes protonated and facilitates osmotic swelling that leads to cargo release into the cytosol, a mechanism known as the proton sponge effect [77]. By this mechanism, the polyhistidine tag facilitated the escape of anti-PSMA aptamer-siRNA chimera from late endosome to cytosol resulting in improved silencing of the target gene [78].

Another straightforward strategy for cytoplasmic delivery is to perform aptamer selection against a cell surface target protein that internalizes to other compartments by receptor-mediated endocytosis. RME facilitates more rapid internalization of the targeting moiety as compared to untargeted complexes. Depending on

the intracellular receptor-dependent or independent trafficking path, the fate of targeting moiety can be controlled, e.g., macromolecules internalized by clathrindependent RME are mostly destined for lysosomal degradation whereas clathrinindependent internalization leads to endosomal accumulation and sorting to a non-degradative path [74]. However, this method can be used against those targets that have previously been characterized, e.g., nucleolin. Nucleolin is a nuclear ribonucleoprotein that is overexpressed in cancer cells where it shuttles among the cell-surface, cytoplasm and the nucleus. AS1411 aptamer, a G-quadruplex forming oligonucleotide, specifically binds nucleolin and displays antiproliferative activity against cancer cells [79]. However, there are conflicting reports regarding nucleolinmediated uptake of AS1411 since one study suggest that it is nucleolin dependent and the other suggests that it is nucleolin independent. Though the mechanism of escape of AS1411 from endosomes to cytoplasm is unclear, it is likely that its interaction with nucleolin, which shuttles between different cellular compartments, plays a role in it [80].
