Drug delivery to the central nervous system is restricted by the blood-brain barrier (BBB). However, with the onset of stroke, the BBB becomes leaky, providing a window of opportunity to passively target the brain. Here, cationic poly(amido amine) (PAMAM) dendrimers of different generations were functionalized with poly(ethylene glycol) (PEG) to reduce cytotoxicity and prolong blood circulation half-life, aiming for a safe in vivo drug delivery system in a stroke scenario. Rhodamine B isothiocyanate (RITC) was covalently tethered to the dendrimer backbone and used as a small surrogate drug as well as for tracking purposes. The biocompatibility of PAMAM was markedly increased by PEGylation as a function of dendrimer generation and degree of functionalization. The PEGylated RITC-modified dendrimers did not affect the integrity of an in vitro BBB model. Additionally, the functionalized dendrimers remained safe when in contact with the bEnd.3 cells and rat primary astrocytes composing the in vitro BBB model after hypoxia induced by oxygen-glucose deprivation. Modification with PEG also decreased the interaction and uptake by endothelial cells of PAMAM, indicating that the transport across a leaky Version: Postprint (identical content as published paper) This is a self-archived document from i3S-Instituto de Investigação e Inovação em Saúde in the University of Porto Open Repository For Open Access to more of our publications, please visit http://repositorio-aberto.up.pt/ A01/00 BBB due to focal brain ischemia would be facilitated. Next, the functionalized dendrimers were tested in contact with red blood cells showing no haemolysis for the PEGylated PAMAM, in contrast to the unmodified dendrimer. Interestingly, the PEG-modified dendrimers reduced blood clotting, which may be an added beneficial function in the context of stroke. The optimized PAMAM formulation was intravenously administered in mice after inducing permanent focal brain ischemia. Twenty-four hours after administration, dendrimers could be detected in the brain, including in neurons of the ischemic cortex. Our results suggest that the proposed formulation has the potential for becoming a successful delivery vector for therapeutic application to the injured brain after stroke reaching the ischemic neurons. Graphical abstract (A) PEG-conjugated PAMAM dendrimers bound with RITC diffusing through a 'leaky' blood brain barrier upon focal cerebral ischemia. (B) Modified PAMAM dendrimer identified in neurons at the site of the brain ischemic injury.

Central nervous system (CNS) disorders encompass a vast spectrum of pathological conditions and represent a growing concern worldwide. Despite the high social and clinical interest in trying to solve these pathologies, there are many challenges to bridge in order to achieve an effective therapy. One of the main obstacles to advancements in this field that has hampered many of the therapeutic strategies proposed to date is the presence of the CNS barriers that restrict the access to the brain. However, adequate brain biodistribution and neuronal cells specific accumulation in the targeted site also represent major hurdles to the attainment of a successful CNS treatment. Over the last few years, nanotechnology has taken a step forward towards the development of therapeutics in neurologic diseases and different approaches have been developed to surpass these obstacles. The versatility of the designed nanocarriers in terms of physical and chemical properties, and the possibility to functionalize them with specific moieties, have resulted in improved neurotargeted delivery profiles. With the concomitant progress in biology research, many of these strategies have been inspired by nature and have taken advantage of physiological processes to achieve brain delivery. Here, the different nanosystems and targeting moieties used to achieve a neuronal delivery reported in the open literature are comprehensively reviewed and critically discussed, with emphasis on the most recent bioinspired advances in the field. Finally, we express our view on the paramount challenges in targeted neuronal delivery that need to be overcome for these promising therapeutics to move from the bench to the bedside.

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