Transport Mechanisms of Squalenoyl-Adenosine Nanoparticles Across the Blood−Brain Barrier Alice Gaudin, † Oya Tagit, ‡ Dunja Sobot, † Sinda Lepetre-Mouelhi, † Julie Mougin, † Thomas F. Martens, §,∥ Kevin Braeckmans, §,∥ Vale ́ rie Nicolas, ⊥ Didier Desmaë le, † Stefaan C. de Smedt, § Niko Hildebrandt, ‡ Patrick Couvreur,* ,† and Karine Andrieux † † Institut Galien Paris-Sud UMR CNRS 8612, Faculty of Pharmacy, and ⊥ Institut d’Innovation The ́ rapeutique, IFR141 ITFM, Faculty of Pharmacy, Universite ́ Paris-Sud, 92296 Châ tenay-Malabry, France ‡ NanoBioPhotonics, Institut d’Electronique Fondamentale, Universite ́ Paris-Sud, 91405 Orsay Cedex, France § Laboratory for General Biochemistry & Physical Pharmacy, Faculty of Sciences, and ∥ Center for Nano- and Biophotonics, Ghent University, 9000 Ghent, Belgium * S Supporting Information ABSTRACT: Drug delivery to the brain is one of the major challenges in the treatment of cerebral diseases and implies extensive understanding of nanomedicine transcytosis path- ways across the blood−brain barrier (BBB). In this study, we investigated the interaction of squalenoyl-adenosine nano- assemblies (SQAd NAs) with human brain endothelial cells, concerning their endocytotic pathway using chemical inhib- itors and nanostructure integrity using Fö rster resonance energy transfer (FRET). Practically, SQAd NAs were labeled with two different organic dyes as a donor−acceptor FRET pair to form FRET SQAd NAs with diameters of ca. 120 nm. Using the human cerebral endothelial cell line, hCMEC/D3, as a well-recognized BBB model, we demonstrated that the NAs were internalized mainly by LDL receptors-mediated endocytosis, then progressively disassembled inside the cells, and finally exocytosed as single molecules. These observations allow explaining the previously described pharmacological efficiency of the SQAd NAs in both a cerebral ischemia model and a spinal cord injury model, confirming that the endothelial cells of the neurovascular unit may represent a very promising therapeutic target for the treatment of certain neurological diseases. ■ INTRODUCTION With approximately 1 billion people worldwide affected by central nervous system (CNS) disorders, CNS drugs represent one of the fastest growing therapeutic segments of the pharmaceutical market 1 and are estimated to account for the strongest medical need of the 21st century. However, CNS drugs in clinical development have a considerably lower probability of entering the market compared to other therapeutics. 2 This is due to the high complexity of the human brain, which leads to a lack of validated biomarkers, to the propensity to cause CNS-mediated side effects and to the presence of the blood−brain barrier (BBB), which prevents the entry of more than 98% of all drugs into the brain parenchyma. 3 The paradox is that more than 99% of the global CNS drug development effort is devoted to CNS drug discovery while less than 1% is devoted to CNS drug delivery, 4 restricting the possibility of new CNS drugs only to lipid-soluble compounds with a molecular weight inferior to 500 Da. Therefore, the use of nanotechnologies has been suggested for delivering drugs into the CNS. 5,6 Indeed, drug nanocarriers such as liposomes, polymeric nanoparticles and solid lipid nanoparticles, may be tailor-made in order to prolong drugs blood circulation and to allow specific cell targeting properties. 7 In this context, we have previously shown that the chemical linkage of the lipid squalene (i.e., ≪squalenoylation≫ ) 8 to adenosine (SQAd) and the subsequent formulation as nanoassemblies (NAs) provided a dramatic pharmacological efficiency in both experimental models of cerebral ischemia in mice and spinal cord injury in rats. 9 Most probably, this effect resulted from an interaction of the NAs or their molecular components with the micro- circulation and the neurovascular unit (NVU), mainly composed by endothelial cells, pericytes, astrocytes endfeet, and neurons, thereby allowing an indirect central effect. 9 In this study, we intended to clarify the interaction of these SQAd NAs with the brain endothelial cells of the BBB, in order to understand by which pathways the NAs are captured by these cells, how they are handled intracellularly and how the transcytosis process occurs. To this aim, the hCMEC/D3 cell line, a well-characterized human brain capillary endothelial cell Received: January 21, 2015 Revised: February 23, 2015 Article pubs.acs.org/cm © XXXX American Chemical Society A DOI: 10.1021/acs.chemmater.5b00267 Chem. Mater. XXXX, XXX, XXX−XXX