Paracellular permeability across epithelial and endothelial cells is, in large part, regulated by apical intercellular junctions also referred to as tight junctions (TJs). These junctions contribute to the spatial definition of different tissue compartments within organisms, separating them from the outside world as well as from inner compartments, with their primary physiological role of maintaining tissue homeostasis. TJs restrict the free, passive diffusion of ions and hydrophilic small molecules through paracellular clefts and are important for appropriate cell polarization and transporter protein localisation, supporting the controlled transcellular diffusion of smaller and larger hydrophilic as well as hydrophobic substances. This traditional diffusion barrier concept of TJs has been challenged lately, owing to a better understanding of the components that are associated with TJs. It is now well-established that mutations in TJ proteins are associated with a range of human diseases and that a change in the membrane fluidity of neighbouring cells can open possibilities for therapeutics to cross intercellular junctions. Nanotechnological approaches, exploiting ultrasound or hyperosmotic agents and permeation enhancers, are the paradigm for achieving enhanced paracellular diffusion. The other widely used transport route of drugs is via transcellular transport, allowing the passage of a variety of pro-drugs and nanoparticle-encapsulated drugs via different mechanisms based on receptors and others. For a long time, there was an expectation that lipidic nanocarriers and polymeric nanostructures could revolutionize the field for the delivery of RNA and protein-based therapeutics across different biological barriers equipped with TJs (e.g., blood–brain barrier (BBB), retina–blood barrier (RBB), corneal TJs, etc.). However, only a limited increase in therapeutic efficiency has been reported for most systems until now. The purpose of this review is to explore the reasons behind the current failures and to examine the emergence of synthetic and cell-derived nanomaterials and nanotechnological approaches as potential game-changers in enhancing drug delivery to target locations both at and across TJs using innovative concepts. Specifically, we will focus on recent advancements in various nanotechnological strategies enabling the bypassing or temporally opening of TJs to the brain and to the retina, and discuss their advantages and limitations.