*3.2.1 Mechanism of cellular uptake*

Cellular uptake refers to the combination of both OD membrane-binding and internalization. It is challenged by numerous barriers, most importantly, the lipophilic nature of the cell membrane, which makes the passage of these large anionic molecules complicated. Several barriers to cellular uptake exist such as the lipophilic cell membrane, through which these large, anionic molecules must pass to reach the

site of action. In cultured cells, the internalization of naked ASOs is generally inefficient, with only a few ASO molecules actually penetrating the cell [21]. Exogenously administered ASOs enter cells *in vitro* by a combination of fluid-phase (pinocytosis), caveolae potocytosis, adsorptive and receptor-mediated endocytosis (**Figure 3**), cellular uptake mechanisms being different depending on the ASO chemical structure. However, this results in a trafficking problem because not all of the internalized ASO will be able to reach their target and interact with it. This is because the majority of internalized ASO is sequestered into endosome or lysosomal compartments [21]. A significant amount of the OD is also compartmentalized within other cellular organelles, such as the Golgi complex and the endoplasmic reticulum [64].

To improve cellular uptake and OD's activity, a range of techniques and transporters have been developed [65, 66]. Simultaneously, the use of these vectors increases the stability of ODs against nuclease digestion and allows the use of lower concentrations of ODs.
