The blood brain barrier (BBB) is a selective biological barrier located at the brain capillaries, that protects the central nervous system (CNS) by monitoring exchanges between blood and brain. The BBB controls and regulates the composition of the CNS environment and it still constitutes the main obstacle for drug delivery to the brain (Weiss N. et al., 2009). The significant scientific and industrial interest in the physiology and pathology of the BBB led to the development of vast number of in vitro BBB models. Even though no “ideal” model exists yet, some of the currently available ones are very useful to investigate permeability, transport mechanisms and cellular and molecular events which occur at the BBB level. New strategies for brain targeted drug delivery exploit endogenously expressed transporters to elicit drug passage across the BBB. Among them, nanoparticles represent a promising tool, since they are biocompatible and biodegradable, and they can be functionalized to target the BBB (De Boer A.G. and Gaillard P.J., 2007) (Beija M et al., 2012; Caruthers S.D. et al., 2007; Moghimi S.M. et al., 2005). In this study we settled in vitro BBB models to identify, with high-throughput screening, the most promising nanoliposomes (NL) for combined BBB crossing and binding of amyloid peptides, for joint therapy and diagnosis of Alzheimer’s disease (AD). Firstly, we characterized two in vitro models of BBB, based on immortalized cell lines of human and rat origin, the hCMEC/D3 and RBE4 cells, respectively. We tested the transendothelial electrical resistance (TEER) and the endothelial permeability (PE) of small hydrophilic compounds: our results, in agreement with data reported in literature, lead us to conclude that these cellular models are suitable for their employment as high-throughput screening tools. Subsequently, we tested NL mono-functionalized with three different peptides, the apolipoproteinE derived peptide (the ApoE monomer (mApoE), amino acids 141-150), its tandem dimer (dApoE) (141-150)2, and the Human Immunodeficency Virus type 1 (HIV-1) transactivator of transcription (TAT) peptide. We evaluated their uptake and PE; we selected the TAT functionalization as the best performing concerning cellular uptake, and the mApoE functionalization when considering both the internalization and PE. Once assessed the dynamics of mono-functionalized NL interactions with endothelial cells, we investigated mApoE- and dApoE-NL loading a curcumin-derivative (Re F. et al., 2011) to bind Aβ. We clearly demonstrated that the mApoE-functionalization allows a better drug cellular internalization, whereas dApoE-NL enhances drug PE at the highest extent. We then considered mApoE- and dApoE-NL exposing the Aβ targeting ligands phosphatidic acid (PA) or cardiolipin (CL), demonstrating that PA-mApoE-NL showed the highest cellular uptake and PE. We also studied TAT-NL exposing curcumin derivative3 (Airoldi C. et al., 2011) for Aβ binding, clearly indicating that TAT functionalization increased cellular uptake and PE of curcumin derivative3-NL. We also studied intracellular fate of NL double functionalized, exposing Aβ targeting ligands, and no co-localization was detected with acidic cellular compartments, suggesting that NL may escape from lysosomal degradative pathway. Taken together, these results indicate that the formulations herein analyzed are suitable tools for brain targeted drug and contrast agent delivery. We suggest further development of mApoE and dApoE-NL entrapped with a drug payload for their employment as BBB endothelial cell or brain targeted drug delivery tools, respectively. We also selected PAmApoE-NL and curcumin derivative3-TAT-NL as promising tools for their employment in combination for AD therapy and diagnosis. Further studies, based also on in vivo experiments, are needed to evaluate NL suitability for clinical exploitation. Finally, we inquired the endocytic mechanisms that mediates the entry of NL in the endothelial cells of BBB. We employed RNA interference technique to down-regulate caveolin1 expression. Our preliminary data suggest that caveolin1 and the related caveolaemediated endocytosis pathway may account for 40% of mApoE-NL cellular uptake. Future directions regard the down-regulation of other proteins specifically involved in different endocytic mechanisms, i.e. clathrin-mediated and adsoprptive endocytosis, in order to assess which endocytic mechanisms may account for ApoE and TAT-NL internalization.
(2012). In vitro blood brain barrier models as a screening tool for brain targeted nanobased drug delivery systems. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2012).
CAVALETTI, GUIDO ANGELO
|Data di pubblicazione:||13-dic-2012|
|Titolo:||In vitro blood brain barrier models as a screening tool for brain targeted nanobased drug delivery systems|
|Settore Scientifico Disciplinare:||BIO/09 - FISIOLOGIA|
|Corso di dottorato:||NEUROSCIENZE - 17R|
|Citazione:||(2012). In vitro blood brain barrier models as a screening tool for brain targeted nanobased drug delivery systems. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2012).|
|Parole Chiave (Inglese):||blood–brain barrier; rat; human; in vitro model; cell culture; brain capillary endothelial cell; immortalized cell line; nanoliposomes; permeability; cell uptake; transporter; Alzheimer's disease (AD)|
|Appare nelle tipologie:||07 - Tesi di dottorato Bicocca post 2009|