Supplementary MaterialsSupplementary Information srep11990-s1. in CNS cells, thus aiding Salinomycin cost the delivery of relevant cargo within their cytosol. We prove this using IgG as a model cargo, thus demonstrating that this combination of appropriate targeting combined with pH-sensitive polymersomes enables the efficient delivery of macromolecules into CNS cells. The fundamental role of the Central Nervous System (CNS, which comprises the brain and spinal cord) in controlling body functions is usually associated with its isolation from the rest of the body. A tight network of membrane barriers controls the transport of nutrients, metabolites and signalling molecules in and out of the CNS, with permeability and trafficking uniquely tailored to the CNS. These barriers include the blood-brain barrier (BBB) at the brain microvascular endothelium, and the blood-cerebrospinal fluid barrier (BCSFB) at the choroid plexus and the arachnoid epithelium1. Of these, the BBB is usually arguably the most important barrier as it allows access to almost all components of the CNS, being the largest in surface area and the one with the shortest diffusion distance to individual cells of the CNS parenchyma1. The BBB is not only an anatomical barrier, but also acts as a metabolic barrier to very precisely control transport between the blood and the CNS. The BBB consists of specialised and highly polarised vascular endothelial cells, which in contrast to peripheral endothelia lack fenestrations, show low expression of immune cell adhesion molecules, and express extremely tight tight junctions that lead to severe restriction of paracellular transport. Brain endothelial cells also control transcellular transport by the expression of specialised molecular transporters at the apical and basolateral membranes, and by limiting vesicular transport via transcytosis to relatively few ligands2. These unique phenotypic functions are the result of the conversation with CNS-resident pericytes3, astrocytes2, microglia and neurons1. Together with the endothelial cells, these cell types form the so-called neurovascular unit. This highly regulated and relatively impermeable barrier is usually a major obstacle for developing new therapeutic approaches to treat neurological diseases4,5,6, and engineering new probes to study the complexity of Salinomycin cost the CNS. One approach to address this problem is usually to develop a carrier that exploits endogenous transcytosis routes to traverse the BBB, enabling the delivery of therapeutics into the CNS without disrupting homeostasis. Transcytosis involves the formation of membrane-bound vesicles around the apical side of endothelia that are quickly moved Salinomycin cost to the basolateral side where the vesicles fuse with the membrane, releasing the cargo within the CNS7. Such a transport mechanism enables the movement of macromolecules, including several proteins and lipoproteins. Furthermore, it is often used by pathogens to gain entry to the CNS8. Achieving transcytosis by targeting endogenous transport systems of the BBB is usually a highly selective and non-invasive delivery mechanism for the CNS, which should be particularly relevant for macromolecular payloads. Several receptors for receptor-mediated transcytosis (RMT) are highly expressed around the endothelial cells that form the BBB, including the low-density lipoprotein receptor-related protein 1 (LRP-1), insulin receptor (IR), transferrin receptor (TfR) and others9,10,11,12. Previous efforts using ligand-functionalised carriers, including solid lipid nanoparticles13, liposomes14, dendrimers15 and micelles16, have been reported to facilitate delivery across the BBB. However, even in the best cases the delivery efficacy has not led to clinical translation, hence more effective strategies to improve CNS delivery are still required. Furthermore, traversing the CNS is not the only challenge associated with designing effective therapeutics. Often the cargo requires delivery into specific CNS sub-compartments, or even entry into CNS resident cells to access their machinery more effectively. Here we use and approaches to examine the combination of transcytosis-targeting motifs with pH-sensitive polymersomes that have been previously demonstrated to facilitate cellular delivery17,18,19,20. We use an established 3D transwell co-culture setup to mimic the Rabbit polyclonal to MBD3 BBB screening Polymersomes including POEGMA-PDPA (EP), PMPC-PDPA (Supplementary Fig. 1a and 1b) and peptide-functionalised EP were prepared via a pH-switch method; this is a bottom-up self-assembly process that can be precisely manipulated, as reported elsewhere35,36. The resulting polymersomes had a mean diameter of 100?nm (Supplementary Fig. 1c) and transmission electron microscopy (TEM) studies confirmed their vesicular morphology (Supplementary Fig. 1d). Further physicochemical characteristics, and their uptake by the mouse brain endothelial cell line bEnd.3, can be found in Supplementary Fig. 1 and Supplementary Fig. 2. The most effective formulations for cellular uptake were further Salinomycin cost tested for transcytosis efficiency. To do so, we employed a 3D BBB model where brain endothelial cells were cultured on collagen-coated trans-well microporous filter inserts.