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Sant'Ambrogio di Torino, Italy

University of Milan, Nanovector S.R.L., University of Pisa and National Research Council Italy | Date: 2010-02-23

The method for preventing and delaying inherited retinal degenerations using serine palmitoyltransferase inhibitors, and compositions which contain them.

Gobbi M.,Mario Negri Institute for Pharmacological Research | Re F.,University of Milan Bicocca | Canovi M.,Mario Negri Institute for Pharmacological Research | Beeg M.,Mario Negri Institute for Pharmacological Research | And 8 more authors.
Biomaterials | Year: 2010

The neurotoxic beta-amyloid peptide (Aβ), formed in anomalous amounts in Alzheimer's disease (AD), is released as monomer and then undergoes aggregation forming oligomers, fibrils and plaques in diseased brains. Aβ aggregates are considered as possible targets for therapy and/or diagnosis of AD. Since nanoparticles (NPs) are promising vehicles for imaging probes and therapeutic agents, we realized and characterized two types of NPs (liposomes and solid lipid nanoparticles, 145 and 76 nm average size, respectively) functionalized to target Aβ1-42 with high affinity. Preliminary immunostaining studies identified anionic phospholipids [phosphatidic acid (PA) and cardiolipin (CL)] as suitable Aβ1-42 ligands. PA/CL-functionalized, but not plain, NPs interacted with Aβ1-42 aggregates as indicated by ultracentrifugation experiments, in which binding reaction occurred in solution, and by Surface Plasmon Resonance (SPR) experiments, in which NPs flowed onto immobilized Aβ1-42. All these experiments were carried out in buffered saline. SPR studies indicated that, when exposed on NPs surface, PA/CL display very high affinity for Aβ1-42 fibrils (22-60 nm), likely because of the occurrence of multivalent interactions which markedly decrease the dissociation of PA/CL NPs from Aβ Noteworthy, PA/CL NPs did not bind to bovine serum albumin. The PA/CL NPs described in this work are endowed with the highest affinity for Aβ so far reported. These characteristics make our NPs a very promising vector for the targeted delivery of potential new diagnostic and therapeutic molecules to be tested in appropriate animal models. © 2010 Elsevier Ltd.

Cambianica I.,University of Milan Bicocca | Bossi M.,University of Milan Bicocca | Gasco P.,Nanovector Srl | Gonzalez W.,Guerbet Res | And 7 more authors.
AIP Conference Proceedings | Year: 2010

Magnetic iron oxide nanoparticles (NPs) are considered for various diagnostic and therapeutic applications in brain including their use as contrast agent for magnetic resonance imaging. In delivery application, the critical step is the transport across cell layers and the internalization of NPs into specific cells, a process often limited by poor targeting specificity and low internalization efficiency. The development of the models of brain endothelial cells and choroidal plexus epithelial cells in culture has allowed us to investigate into these mechanisms. Our strategy is aimed at exploring different routes to the entrapment of iron oxide NPs in these brain related cells. Here we demonstrated that not only cells endowed with a good phagocytic activity like activated macrophages but also endothelial brain capillary and choroidal plexus epithelial cells do internalize iron oxide NPs. Our study of the intracellular trafficking of NPs by TEM, and confocal microscopy revealed that NPs are mainly internalized by the endocytic pathway. Iron oxide NPs were dispersed in water and coated with 3,4-dihydroxyl-L-phenylalanine (L-DOPA) using standard procedures. Magnetic lipid NPs were prepared by NANOVECTOR: water in oil in water (W/O/W) microemulsion process has been applied to directly coat different iron based NPs by lipid layer or to encapsulate them into Solid Lipid Nanoparticles (SLNs). By these coating/loading the colloidal stability was improved without strong alteration of the particle size distribution. Magnetic lipid NPs could be reconstituted after freeze drying without appreciable changes in stability. L-DOPA coated NPs are stable in PBS and in MEM (Modified Eagle Medium) medium. The magnetic properties of these NPs were not altered by the coating processes. We investigated the cellular uptake, cytotoxicity, and interaction of these NPs with rat brain capillary endothelial (REB4) and choroidal plexus epithelial (Z310) cells. By means of widefield, confocal microscopy and flow cytometry we studied the cell uptake of magnetic SLNs derivatized with a fluorescent reporter molecule and of L-DOPA-TRITC coated NPs. Inhibition of the caveolae-mediated pathway by preincubation with filipin and nystatin did not modify the cellular uptake of these NPs in both cell lines. Furthermore a mild decrease of the NPs cell uptake was obtained after chlorpromazine and NaN3 pretreatment, which interferes with clathrin and energy-dependent endocytosis, and cytochalasin and amiloride pretreatment which interfere with macropinocytosis. NPs particle size as such can strongly affect the efficiency of cellular uptake and the mode of endocytosis. Considering that our L-DOPA and magnetic SLNs display a medium hydrodynamic size of 120 nm with a polydispersity index of 0.3, we can assume that the cell uptake process of these NPs may develop, depending the particle size, both via clathrin mediated endocytosis and macropinocytosis and only to less extent via the pathway of caveolae-mediated endocytosis. Taken together these results let us to conclude that SLNs iron loaded and iron based L-DOPA coated NPs are internalized into brain endothelial and choroidal plexus epithelial cells and this might provide the first step of an intracellular trafficking to transport these NPs between blood and brain. © 2010 American Institute of Physics.

Priano L.,Istituto Auxologico Italiano | Priano L.,University of Turin | Zara G.P.,University of Turin | El-Assawy N.,Istituto Auxologico Italiano | And 10 more authors.
European Journal of Pharmaceutics and Biopharmaceutics | Year: 2011

Intrathecal baclofen administration is the reference treatment for spasticity of spinal or cerebral origin, but the risk of infection or catheter dysfunctions are important limits. To explore the possibility of alternative administration routes, we studied a new preparation comprising solid lipid nanoparticles (SLN) incorporating baclofen (baclofen-SLN). We used SLN because they are able to give a sustained release and to target the CNS. Wistar rats were injected intraperitoneally with baclofen-SLN or baclofen solution (baclofen-sol group) at increasing dosages. At different times up to 4 h, efficacy was tested by the H-reflex and two scales evaluating sedation and motor symptoms due to spinal lesions. Rats were killed and baclofen concentration determined in blood and tissues. Physiological solution or unloaded SLN was used as controls. After baclofen-SLN injection, the effect, consisting in a greater and earlier reduction of the H/M ratio than baclofen-sol group and controls, was statistically significant from a dose of 5 mg/kg and was inversely correlated with dose. Clinical effect of baclofen-SLN on both the behavioral scales was greater than that of baclofen-sol and lasted until 4th hour. Compared with baclofen-sol, baclofen-SLN produced significantly higher drug concentrations in plasma from 2nd hour until 4th hour with a linear decrement and in the brain at all times. In conclusion, our study demonstrated the efficacy of a novel formulation of baclofen, which exploits the advantages of SLN preparations. However, for clinical purposes, high baclofen concentrations in brain tissue and sedation may be unwanted effects, requiring further studies and optimization of dosages. © 2011 Elsevier B.V. All rights reserved.

Panariti A.,University of Milan Bicocca | Rivolta I.,University of Milan Bicocca | Lettiero B.,University of Milan Bicocca | Chirico G.,University of Milan Bicocca | And 2 more authors.
AIP Conference Proceedings | Year: 2010

Solid Lipid Nanoparticles (SLN) are well defined nanocarriers that reach the close proximity of the cell membrane and bring their content into the cell. We studied the distribution of the SLN within the cell cytoplasm trying to understand the internalization mechanism of these nanoparticles. Alveolar epithelial lung and mouse fibroblast cells were incubated with SLN. Our data suggest that SLN may be a compatible tool but still deserve further investigation. © 2010 American Institute of Physics.

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