RESUMO
PURPOSE: We investigated the ability of amyloid-ß-targeting liposomes, decorated with an anti-transferrin receptor antibody, to cross the blood-brain barrier (BBB), comparing two antibody ligation techniques. METHODS: Fluorescent or radiolabeled liposomes composed of sphingomyelin/cholesterol and containing phosphatidic acid, known to bind amyloid-ß, were further functionalized with the anti-transferrin receptor antibody RI7217. Two different techniques were used to attach RI7217 to the liposomes surface: biotin/streptavidin linkage or thiol-maleimide covalent ligation. Surface plasmon resonance (SPR) and immunoblotting were employed to assess the nanoparticles' binding performances. Confocal microscopy and radiochemical techniques were used for uptake and permeability studies on an in vitro BBB model made of human brain capillary endothelial cells hCMEC/D3. RESULTS: Immunoblotting experiments showed that RI7217-functionalized liposomes bind to transferrin receptor independently of the procedure employed to ligate their surface with the antibody, while SPR experiments showed a slightly higher affinity for covalently functionalized nanoliposomes. The functionalization with RI7217 did not affect the liposomes' affinity for amyloid-ß. The functionalization of liposomes with RI7217, independently of the ligation procedure, gave higher values of uptake and permeability across the barrier model in comparison to the nondecorated ones, without cell monolayer alterations. Of note, the best performing particles were those covalently coupled with the antibody. The ratios of the two radiolabeled lipids ((3)H-sphingomyelin and (14)C-phosphatidic acid) present in the liposome bilayer were found to be similar in the apical and in the basolateral compartments of the barrier model, suggesting that liposomes were transported intact across the cell monolayer. Confocal experiments showed no co-localization of RI7217-liposomes with early/late endosomes or early lysosomes. CONCLUSION: Our results suggest that RI7217 promotes the in vitro barrier crossing of liposomes containing phosphatidic acid, targeting the Alzheimer's disease amyloid-ß peptide. Moreover, for the first time, we prove herein the superior efficiency of covalent coupling of RI7217 versus biotin/streptavidin ligation to facilitate liposomes in overcoming the BBB in vitro.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Portadores de Fármacos/farmacocinética , Lipossomos/farmacocinética , Modelos Biológicos , Peptídeos beta-Amiloides , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Endotélio Vascular/citologia , Humanos , Lipossomos/química , Lipossomos/farmacologia , Ácidos Fosfatídicos/química , Receptores da Transferrina/imunologia , Ressonância de Plasmônio de SuperfícieRESUMO
Nanoliposomes containing phosphatidic acid or cardiolipin are able to target in vitro with very high affinity amyloid-ß (Aß), a peptide whose overproduction and progressive aggregation in the brain play a central role in the pathogenesis of Alzheimer's disease. However, the presence of the blood-brain barrier (BBB) severely limits the penetration of either drugs or drug vehicles (nanoparticles) to the brain. Therefore, there is a need to develop and design approaches specifically driving nanoparticles to brain in a better and effective way. The aim of the present investigation is the search of a strategy promoting the interaction of liposomes containing acidic phospholipids with brain capillary endothelial cells, as a first step toward their passage across the BBB. We describe the preparation and physical characterization of nano-sized liposomes decorated with peptides derived from apolipoprotein E and characterize their interaction with human immortalized brain capillary cells cultured in vitro (hCMEC/D3). For this purpose, we synthesized two ApoE-derived peptides (the fragment 141-150 or its tandem dimer) containing a cysteine residue at the C-terminus and decorated NL by exploiting the cysteine reaction with a maleimide-group on the nanoparticle surface. NL without ApoE functionalization did not show either relevant membrane accumulation or cellular uptake, as monitored by confocal microscopy using fluorescently labeled nanoliposomes or quantifying the cell-associated radioactivity of isotopically labeled nanoliposomes. The uptake of nanoliposomes by cell monolayers was enhanced by ApoE-peptide-functionalization, and was higher with the fragment 141-150 than with its tandem dimer. The best performance was displayed by nanoliposomes containing phosphatidic acid and decorated with the ApoE fragment 141-150. Moreover, we show that the functionalization of liposomes containing acidic phospholipids with the ApoE fragment 141-150 scarcely affects their reported ability to bind Aß peptide in vitro. These are important and promising features for the possibility to use these nanoliposomes for the targeting of Aß in the brain districts.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Apolipoproteínas E/metabolismo , Células Endoteliais/metabolismo , Lipossomos/metabolismo , Nanopartículas/química , Doença de Alzheimer , Apolipoproteínas E/química , Linhagem Celular Transformada , Humanos , Lipossomos/química , Lipossomos/farmacocinética , Microscopia Confocal , Fosfolipídeos , Ligação Proteica , TrítioRESUMO
A promising strategy to enhance blood-brain barrier penetration by drugs is the functionalization of nanocarriers with uptake-facilitating ligands. We studied the cellular uptake, by cultured RBE4 brain capillary endothelial cells, of nanoliposomes (NLs) covalently coupled with monomer or tandem dimer of apolipoprotein E (ApoE)-derived peptides (residues 141-150), at various densities. NLs without functionalization did not show either relevant membrane accumulation or cellular uptake, as monitored by confocal microscopy and quantified by fluorescence-activated cell sorting. Functionalization with peptides mediated an efficient NLs uptake that increased with peptide density; NLs carrying monomeric peptide performed the best. Moreover, we studied the ability of ApoE-NLs to enhance the transport of a drug payload through a RBE4 cell monolayer. The permeability of a tritiated curcumin derivative was enhanced after its entrapment into ApoE-NLs, in particular those functionalized with the dimer (+83% with respect to free drug, P < 0.01). Thus, these NLs appear particularly suitable for implementing further strategies for drug brain targeting.
Assuntos
Apolipoproteínas E/química , Barreira Hematoencefálica/metabolismo , Sistemas de Liberação de Medicamentos , Nanopartículas/administração & dosagem , Animais , Transporte Biológico , Encéfalo/metabolismo , Linhagem Celular , Curcumina/farmacocinética , Células Endoteliais/metabolismo , Citometria de Fluxo , Humanos , Lipossomos , Microscopia Confocal , Permeabilidade , RatosRESUMO
After denervation of adult rat abdominal muscles, the postsynaptic apparatus of neuromuscular junctions (NMJs) retains its original architecture and clustering of acetylcholine receptors (AChRs). When descending fibers of the spinal cord are surgically diverted to this muscle by a nerve grafting procedure, supraspinal glutamatergic neurons can innervate muscle fibers and restore motor function; the newly formed NMJs switch from a cholinergic to a glutamatergic-type synapse. We show here that regenerating nerve endings contact the fibers in an area occupied by cholinergic endplates. These NMJs are morphologically indistinguishable from those in controls, but they differ in the subunit composition of AChRs. Moreover, by immunofluorescence and immunoelectron microscopy, new NMJs express glutamatergic synapse markers. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1 partially colocalizes with AChRs, and vesicular glutamate transporter 2 is localized in the presynaptic compartment. Immunoprecipitation analysis of membranes from reinnervated muscle showed that AMPA receptor subunits GluR1 and GluR2 coimmunoprecipitate with rapsyn, the AChR-anchoring protein at the NMJ. Taken together, these results indicate that cholinergic endplates can be targeted by new glutamatergic projections and that the clustering of AMPA receptors occurs there.
Assuntos
Ácido Glutâmico/metabolismo , Placa Motora/fisiologia , Músculo Esquelético/fisiologia , Regeneração Nervosa/fisiologia , Junção Neuromuscular/fisiologia , Receptores Colinérgicos/metabolismo , Animais , Imunofluorescência , Imunoprecipitação , Masculino , Microscopia Imunoeletrônica , Placa Motora/ultraestrutura , Proteínas Musculares/metabolismo , Músculo Esquelético/inervação , Músculo Esquelético/ultraestrutura , Junção Neuromuscular/ultraestrutura , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Wistar , Receptores de AMPA/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismoRESUMO
The large majority of excitatory synapses are located on dendritic spines which are discrete membrane protrusions present on neuronal dendrites. Interestingly the highly heterogeneous morphology of dendritic spines is thought to be the morphological basis for synaptic plasticity associated to learning and memory formation. Indeed dendritic spines structure is regulated by molecular mechanisms that are fine tuned and adjusted according to level and direction of synaptic activity, development, specific brain region, and different experimental behavioral conditions. This supports the idea that reciprocal changes between the structure and function of spines impact both local and global integration of signals within dendrites. An increasing number of proteins have been found to be morphogens for dendritic spines and provided new insights into the molecular mechanisms regulating spine formation and morphology. Thus determining the mechanisms that regulate spine formation and morphology is essential for understanding the cellular changes that underlie learning and memory in normal and pathological conditions.