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Introduction The blood-brain barrier (BBB) plays an important role in maintaining the homeostasis of the central nervous system and in protecting the brain from potentially harmful endogenous and exogenous compounds. Nevertheless it represents also the major obstacle for the diagnosis and therapy of brain diseases. One of the most promising strategies to overcome the limited BBB penetration of drugs and contrast agents is based on nanoparticles (NP). Lipid based NP, basically liposomes and solid lipid nanoparticles (SLN), have several advantages in terms of biocompatibility, non-immunogenicity, non-toxicity; they can be used as carrier systems [1], and they have a high blood circulation residence time [2]. Moreover their surface can be easily modified with ligands which mediate a site-specific targeting. Goal of the work The objective of present investigation was to study the effect of surface characteristics of SLN covalently coupled with the monomer of ApoE-residues (141-150) (mApoE-SLN) in promoting BBB crossing and brain targeting using both in vitro and in vivo models. Methods Radiolabelled or fluorescent dye-loaded SLN, covalently coupled by DSPE-PEG(2000)-Maleimide with the monomer of ApoE-residues (141-150) [3] and functionalized with phosphatidic acid (Aβ ligands) [4], were used in the present work and produced by Nanovector s.r.l. (Torino, Italy). In vitro evaluations were performed using cultured human cerebral microvascular endothelial cells (hCMEC/D3) obtained from Institute Cochin (INSERM, Paris, France). SLN cell uptake was monitored by confocal-laser-scanning microscopy and cell-associated fluorescence was quantified by FACS analysis. Radiochemical technique was used in order to assess the ability of ApoE monomer to enhance SLN transcellular transport across the hCMEC/D3 BBB model [5]. The in vivo biodistribution of SLN, loaded with DiR (near-infrared fluorescent cyanine dye), was evaluated by means of Fluorescent Microscopy Tomography (FMT 1500, Perkin Elmer). BALB/c male mice were intravenous (IV), intratracheal (IT) or intraperitoneal (IP) administered with 50 ul of SLN formulation and tomographic data analyses were achieved using the TrueQuant software supplied (Perkin Elmer). The total amount (in picomoles) of fluorophore in the brain region was calculated by the provided software using previously generated standards of the appropriate dye [6]. Results and Discussion We demonstrated that surface functionalization of SLN with ApoE monomer plays a major role in promoting their cellular uptake within hCMEC/D3. Cell associated fluorescence was about two-fold higher in presence of SLN-mApoE compared to unfunctionalized SLN (SLN-cys) and the same trend was observed by CLSM analysis. The ability of SLN to cross the in vitro hCMEC/D3 BBB model was assessed using dual-radiolabelled formulations. With respect to SLN-cys, the presence of monomer ApoE significantly enhanced their permeability through the cell monolayer; moreover PE values obtained with the two radiotracers were equivalent for the same SLN formulation, and about 6-fold higher for SLN-mApoE (Fig. 1). These results suggest that, at least at the dose tested, SLN cross intact the cell monolayer. In vivo results confirmed the role of monomer ApoE in sustaining the delivery of SLN to the central nervous system. In particular we demonstrated that, compared to the most common routes for drug administration (IP and IV injections), IT instillation represents the best method to guarantee the biodistribution of SLN-mApoE in the brain district and to favour their retention up to 24 hours after the administration (Fig. 2).Bronchoalveolar lavage fluid (BALF) analysis does not evidence any pro-inflammatory reaction in lungs of SLN-mApoE IT-treated mice with no alteration of the alveolar-capillary barrier permeability. Conclusions The results here obtained suggest that the SLN formulation herein analysed could represent a suitable tool for sustaining drug delivery to the brain.

Diagnosis and therapy of brain diseases are often compromised by the difficulty to cross the blood brain barrier (BBB). Recently, the emerging field of nanotechnology has generated new promises to solve this problem. Nanoparticles (NPs) have several advantages in terms of biocompatibility, non-immunogenicity, non-toxicity and they can be functionalized to carry imaging agents and/or drugs, and to enhance theblood circulation residence time. Finally, the NPs surface can be modified with specific ligands in order to achieve site-specific delivery and successful penetration of the BBB. The objective of present investigation was to study the effect of surface characteristics of solid lipid nanoparticles (SLN) covalently coupled with the monomer of ApoE-residues (141-150) on cellular uptake in brain capillary endothelial cells. Radiolabelled and fluorescent (fluoroprobe strictly associated to SLN) have been used to evaluate the transcellular transport in in vitro BBB model based on human cerebral microvascular endothelial cells (hCMEC/D3). SLN made of tripalmitin, loaded with different fluorescent dyes (Bodipy, Tritc and Texas Red) and functionalized with phosphatidic acid (Aβ ligands) and DSPE-PEG(2000)-Maleimide have been investigated. SLN uptake was monitored byconfocal-laser-scanning microscopy and quantified by radiochemical techniques. The peptide mediated an efficient cellular uptake of SLN. SLN without surface-located peptide displayed less membrane accumulation and cellular uptake. In order to assess the ability of ApoE-SLN to enhance their transcellular transport, we studied the permeability through an in vitro BBB model. With respect to the un-functionalized SLN, the ApoE-SLN significantly enhanced their cellular uptake and permeability through the cell monolayer (PE = 0.6 • 10-5 cm/min vs PE = 6.95 • 10-5 cm/min, respectively; Student's t-test, p value. References: 1. Priano L, Zara GP, El-Assawy N, Cattaldo S, Muntoni E, Milano E, Serpe L, Musicanti C, Pérot C, Gasco MR, Miscio G, Mauro A. Baclofen-loaded solid lipid nanoparticles: preparation, electrophysiological assessment of efficacy, pharmacokinetic and tissue distribution in rats after intraperitoneal administration. Eur J Pharm Biopharm. 2011 Sep;79(1):135-41. doi: 10.1016/j.ejpb.2011.02.009. Epub 2011 Feb 23. Pub Med PMID: 21352914. 2. Gasco MR, Priano L, Zara GP. Chapter 10 - Solid lipid nanoparticles and microemulsions for drug delivery The CNS. Prog Brain Res. 2009;180:181-92. doi:10.1016/S0079-6123(08)80010-6. Epub 2009 Dec 8. Review. PubMed PMID: 20302835. 3. Gobbi M, Re F, Canovi M, Beeg M, Gregori M, Sesana S, Sonnino S, Brogioli D, Musicanti C, Gasco P, Salmona M, Masserini ME. Lipid-based nanoparticles with high binding affinity for amyloid-beta1-42 peptide. Biomaterials. 2010 Sep;31(25):6519-29. doi: 10.1016/j.biomaterials. 2010.04.044. Pub Med PMID:20553982. 4. Re F, Cambianica I, Zona C, Sesana S, Gregori M, Rigolio R, La Ferla B, Nicotra F, Forloni G, Cagnotto A, Salmona M, Masserini M, Sancini G. Functionalization of liposomes with ApoE-derived peptides at different density affects cellular uptake and drug transport across a blood-brain barrier model. Nanomedicine. 2011 Oct;7(5):551-9. Epub 2011 May 20. Pub Med PMID: 21658472. 5. Re F, Cambianica I, Sesana S, Salvati E, Cagnotto A, Salmona M, Couraud PO, Moghimi SM, Masserini M, Sancini G. Functionalization with ApoE-derived peptides enhances the interaction with brain capillary endothelial cells of nanoliposomes binding amyloid-beta peptide. J Biotechnol. 2010 Dec 20;156(4):341-6. Epub 2011 Jul 6. Pub Med PMID: 21763360. Acknowledgement / funding: The research leading to these results received funding from the European Community’s Seventh Framework Program (FP7/2007-2013) under grant agreement n° 212043 (NAD). We thank Pierre-Olivier Couraud for providing the hCMEC/D3 cells.

Solid lipid nanoparticles (SLN) are colloidal drug delivery systems characterized by higher entrapment efficiency, good scalability of the preparation process and increased sustained prolonged release of the payload compared to other nanocarriers. The possibility to functionalize the surface of SLN with ligands to achieve a site specific targeting makes them attractive to overcome the limited blood-brain barrier (BBB) penetration of therapeutic compounds. SLN are prepared for brain targeting by exploiting the adaptability of warm microemulsion process for the covalent surface modification with an Apolipoprotein E-derived peptide (SLN-mApoE). Furthermore, the influence of the administration route on SLN-mApoE brain bioavailability is here evaluated. SLN-mApoE are able to cross intact a BBB in vitro model. The pulmonary administration of SLN-mApoE is related to a higher confinement in the brain of Balb/c mice compared to the intravenous and intraperitoneal administration routes, without inducing any acute inflammatory reaction in the lungs. These results promote the pulmonary administration of brain-targeted SLN as a feasible strategy for improving brain delivery of therapeutics.