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Titanium dioxide nanoparticles (TiO2 NPs) are one of the main sources of the nanoparticulate matter exposure to humans. Although several studies have demonstrated their potential toxic effects, the real nature of the correlation between NP properties and their interaction with biological targets is still far from being fully elucidated. Here, engineered TiO2 NPs with various geometries (bipyramids, plates, and rods) have been prepared, characterized and intravenously administered in healthy mice. Parameters such as biodistribution, accumulation, and toxicity have been assessed in the lungs and liver. Our data show that the organ accumulation of TiO2 NPs, measured by ICP-MS, is quite low, and this is only partially and transiently affected by the NP geometries. The long-lasting permanence is exclusively restricted to the lungs. Here, bipyramids and plates show a higher accumulation, and interestingly, rod-shaped NPs are the most toxic, leading to histopathological pulmonary alterations. In addition, they are also able to induce a transient increase in serum markers related to hepatocellular injury. These results indicate that rods, more than bipyramidal and spherical geometries, lead to a stronger and more severe biological effect. Overall, small physico-chemical differences can dramatically modify both accumulation and safety.

Evidence that Huntington's disease (HD) is characterized by impaired cholesterol biosynthesis in the brain has led to strategies to increase its level in the brain of the rapidly progressing R6/2 mouse model, with a positive therapeutic outcome. Here we tested the long-term efficacy of chronic administration of cholesterol to the brain of the slowly progressing zQ175DN knock-in HD mice in preventing (“early treatment”) or reversing (“late treatment”) HD symptoms. To do this we used the most advanced formulation of cholesterol loaded brain-permeable nanoparticles (NPs), termed hybrid-g7-NPs-chol, which were injected intraperitoneally. We show that one cycle of treatment with hybrid-g7-NPs-chol, administered in the presymptomatic (“early treatment”) or symptomatic (“late treatment”) stages is sufficient to normalize cognitive defects up to 5 months, as well as to improve other behavioral and neuropathological parameters. A multiple cycle treatment combining both early and late treatments (“2 cycle treatment”) lasting 6 months generates therapeutic effects for more than 11 months, without severe adverse reactions. Sustained cholesterol delivery to the brain of zQ175DN mice also reduces mutant Huntingtin aggregates in both the striatum and cortex and completely normalizes synaptic communication in the striatal medium spiny neurons compared to saline-treated HD mice. Furthermore, through a meta-analysis of published and current data, we demonstrated the power of hybrid-g7-NPs-chol and other strategies able to increase brain cholesterol biosynthesis, to reverse cognitive decline and counteract the formation of mutant Huntingtin aggregates. These results demonstrate that cholesterol delivery via brain-permeable NPs is a therapeutic option to sustainably reverse HD-related behavioral decline and neuropathological signs over time, highlighting the therapeutic potential of cholesterol-based strategies in HD patients. Data availability: This study does not include data deposit

Supplementing brain cholesterol is emerging as a potential treatment for Huntington's disease (HD), a genetic neurodegenerative disorder characterized, among other abnormalities, by inefficient brain cholesterol biosynthesis. However, delivering cholesterol to the brain is challenging due to the blood-brain barrier (BBB), which prevents it from reaching the striatum, especially, with therapeutically relevant doses. Here we describe the distribution, kinetics, release, and safety of novel hybrid polymeric nanoparticles made of PLGA and cholesterol which were modified with an heptapeptide (g7) for BBB transit (hybrid-g7-NPs-chol). We show that these NPs rapidly reach the brain and target neural cells. Moreover, deuterium-labeled cholesterol from hybrid-g7-NPs-chol is released in a controlled manner within the brain and accumulates over time, while being rapidly removed from peripheral tissues and plasma. We confirm that systemic and repeated injections of the new hybrid-g7-NPs-chol enhanced endogenous cholesterol biosynthesis, prevented cognitive decline, and ameliorated motor defects in HD animals, without any inflammatory reaction. In summary, this study provides insights about the benefits and safety of cholesterol delivery through advanced brain-permeable nanoparticles for HD treatment.

Avidin-Nucleic-Acid-NanoASsemblies (ANANAS) possess natural tropism for the liver and, when loaded with dexamethasone, reduce clinical progression in an autoimmune hepatitis murine model. Here, we investigated the linker chemistry (hydrazide-hydrazone, Hz-Hz, or carbamate hydrazide-hydrazone, Cb-Hz bond) and length (long, 5 kDa PEG, or short, 5-6 carbons) in biotin-dexamethasone conjugates used for nanoparticle decoration through in vitro and in vivo studies. All four newly synthesized conjugates released the drug at acidic pH only. In vitro, the Hz-Hz and the PEG derivatives were less stable than the Cb-Hz and the short chain ones, respectively. Once injected in healthy mice, dexamethasone location in the PEGylated ANANAS outer layer favors liver penetration and resident macrophages uptake, while drug Hz-Hz, but not Cb-Hz, short spacing prolongs drug availability. In conclusion, the tight modulation of ANANAS decoration can significantly influence the host interaction, paving the way for the development of steroid nanoformulations suitable for different pharmacokinetic profiles.

Aim: We investigated the use of cellulose nanocrystals (CNCs) as drug nanocarriers combining an anti-osteoporotic agent, alendronate (ALN), and an anti-cancer drug, doxorubicin (DOX). Materials & methods: CNC physicochemical characterization, in vivo imaging coupled with histology and in vitro uptake and toxicity assays were carried out. Results: In vivo CNC-ALN did not modify bone tropism and lung penetration, whereas its liver and kidney accumulation was slightly higher compared with CNCs alone. In vitro studies showed that CNC-ALN did not impair ALN's effect on osteoclasts, whereas CNC-DOX confirmed the therapeutic potential against bone metastatic cancer cells. Conclusions: This study provides robust proof of the potential of CNCs as easy, flexible and specific carriers to deliver compounds to the bone.

One of the goals of the pharmaceutical sciences is the amelioration of targeted drug delivery. In this context, nanocarrier-dependent transportation represents an ideal method for confronting a broad range of human disorders. In this study, we investigated the possibility of improving the selective release of the anti-cancer drug paclitaxel (PTX) in the gastro-intestinal tract by encapsulating it into the biodegradable nanoparticles made by FDA-approved poly(lactic-co-glycolic acid) (PLGA) and coated with polyethylene glycol to improve their stability (PLGA-PEG-NPs). Our study was performed by combining the synthesis and characterization of the nanodrug with in vivo studies of pharmacokinetics after oral administration in mice. Moreover, fluorescent PLGA-nanoparticles (NPs), were tested both in vitro and in vivo to observe their fate and biodistribution. Our study demonstrated that PLGA-NPs: (1) are stable in the gastric tract; (2) can easily penetrate inside carcinoma colon 2 (CaCo2) cells; (3) reduce the PTX absorption from the gastrointestinal tract, further limiting systemic exposure; (4) enable PTX local targeting. At present, the oral administration of biodegradable nanocarriers is limited because of stomach degradation and the sink effect played by the duodenum. Our findings, however, exhibit promising evidence towards our overcoming these limitations for a more specific and safer strategy against gastrointestinal disorders.

Steroids are the standard therapy for autoimmune hepatitis (AIH) but the long-lasting administration is hampered by severe side effects. Methods to improve the tropism of the drug toward the liver are therefore required. Among them, conjugation to nanoparticles represents one possible strategy. In this study, we exploited the natural liver tropism of Avidin-Nucleic-Acid-Nano-Assemblies (ANANAS) to carry dexamethasone selectively to the liver in an AIH animal model. An acid-labile biotin-hydrazone linker was developed for reversible dexamethasone loading onto ANANAS. The biodistribution, pharmacokinetics and efficacy of free and ANANAS-linked dexamethasone (ANANAS-Hz-Dex) in healthy and AIH mice were investigated upon intraperitoneal administration. In ANANAS-treated animals, the free drug was detected only in the liver. Super-resolution microscopy showed that nanoparticles segregate inside lysosomes of liver immunocompetent cells, mainly involved in AIH progression. In agreement with these observational results, chronic low-dose treatment with ANANAS-Hz-Dex reduced the expression of liver inflammation markers and, in contrast to the free drug, also the levels of circulating AIH-specific autoantibodies. These data suggest that the ANANAS carrier attenuates AIH-related liver damage without drug accumulation in off-site tissues. The safety and biodegradability of the ANANAS carrier make this formulation a promising tool for the treatment of autoimmune liver disorders.

© 2018, United States & Canadian Academy of Pathology. HIV-associated neurocognitive disorder in HIV patients substantially reduces their quality of life. We previously showed that the HIV matrix protein, p17 could stimulate lymph-angiogenesis in vitro potentially contributing to lymphoma tumour growth and in addition is associated with vascular activation in neuro-degenerating brain tissue; here, therefore, we have investigated the detailed molecular mechanisms of this action. We performed in vitro cell culture, angiogenesis experiments, phospho-protein microarrays and Western blotting to identify cellular signalling induced by p17 within human brain endothelial cells (HbMEC), and inhibitor studies to block p17-induced vascular growth. We also characterised the effects of hippocampal CA1 injection of p17 on epidermal growth factor receptor-1 (EGFR1) expression linked to our murine model of dementia. p17 strongly induced angiogenesis of HbMEC (migration, tube formation and spheroid growth). p17 concomitantly increased phosphorylation of EGFR1 as well as down-stream intermediates ERK1/2, FAK, PLC-γ and PKC-β whilst an inhibitor peptide of EGFR, blocked cell signalling and angiogenesis. Finally, Mice that showed reduced cognitive function and behavioural deficiencies after p17 injection, demonstrated that p17 localised in cortical microvessels and also neurones many of which stained positive for p-EGFR1 by histology/IHC. This work provides strong support that p17 may be involved in initiating and/or perpetuating vascular tissue pathophysiology associated with comorbidity in HIV patients.

Human immunodeficiency virus type-1 (HIV-1)-Associated neurocognitive disorder (HAND) remains an important neurological manifestation that adversely affects a patient's quality of life. HIV-1 matrix protein p17 (p17) has been detected in autoptic brain tissue of HAND individuals who presented early with severe AIDS encephalopathy. We hypothesised that the ability of p17 to misfold may result in the generation of toxic assemblies in the brain and may be relevant for HAND pathogenesis. A multidisciplinary integrated approach has been applied to determine the ability of p17 to form soluble amyloidogenic assemblies in vitro. To provide new information into the potential pathogenic role of soluble p17 species in HAND, their toxicological capability was evaluated in vivo. In C. elegans, capable of recognising toxic assemblies of amyloidogenic proteins, p17 induces a specific toxic effect which can be counteracted by tetracyclines, drugs able to hinder the formation of large oligomers and consequently amyloid fibrils. The intrahippocampal injection of p17 in mice reduces their cognitive function and induces behavioral deficiencies. These findings offer a new way of thinking about the possible cause of neurodegeneration in HIV-1-seropositive patients, which engages the ability of p17 to form soluble toxic assemblies.

There is evidence that Tetracyclines are potentially useful drugs to treat prion disease, the fatal neurodegenerative disease in which cellular prion proteins change in conformation to become a disease-specific species (PrPSc). Based on an in vitro anti-fibrillogenesis test, and using the peptide PrP106-126 in the presence of tetracycline and 14 derivatives, we carried out a three-dimensional quantitative structure-activity relationship (3D-QSAR) study to investigate the stereoelectronic features required for anti-fibrillogenic activity. A preliminary variable reduction technique was used to search for grid points where statistical indexes of interaction potential distributions present local maximum (or minimum) values. Variable selection genetic algorithms were then used to search for the best 3D-QSAR models. A 6-variable model showed the best predictability of the anti-fibrillogenic activity that highlighted the best tetracycline substitution patterns: hydroxyl group presence in positions 5 and 6, electrodonor substituents on the aromatic D-ring, alkylamine substituent at the amidic group in position 2 and non-epi configuration of the NMe2 group. © Springer-Verlag 2008.

Extensive molecular dynamic simulations (c.ca 240 ns) have been used to investigate the conformational behavior of PrP106-126 prion peptide in four different environments (water, dimethyl sulfoxide, hexane, and trifluoroethanol) and under both neutral and acidic conditions. The conformational polymorphism of PrP106-126 in solution observed in the simulations supports the role of this fragment in the structural transition of the native to the abnormal form of prion protein in response to changes in the local environmental conditions. The peptide in solution is primarily unstructured. The simulations show an increased presence of helical structure in an apolar solvent, in agreement with the results from circular dichroism spectroscopy. In water solution, b-sheet elements were observed between residues 108-112 and either residues 115-121 or 121-126. An a-b transition was observed under neutral conditions. In DMSO, the peptide adopted an extended conformation, in agreement with nuclear magnetic resonance experiments.

Tetracycline (TC) and its derivatives have recently been proposed as a new class of antagonists in prion diseases as they prevent the aggregation of prion protein peptides and their acquisition of protease resistance in vitro and in vivo. Looking for relationships between conformational flexibility and biological activity, we searched for a geometrical pharmacophore by investigating, in aqueous solution, the conformational behavior of 15 TCs in both the zwitterionic and the anionic forms. For TC similar conformational flexibility was found for the two forms and two main conformational families were detected, an extended and a folded conformation characterized by different intramolecular hydrogen-bond networks. On comparing the Molecular Mechanics results with the ab initio ones and the experimental evidence, it can be seen that the conformational behavior of TC is reasonably well predicted by the MM2 force field, whereas the conformational energies provided by the Amber force field are unreliable. The conformational analysis of the other TC derivatives was then performed by the MM2 force field. As a result, their conformational behavior was similar to that observed for TC itself. Despite the hydronaphthacene moiety's conformational flexibility, no geometrical pharmacophore was found among the TCs, i.e. properties other than geometrical ones should play a crucial role in determining their anti-fibrillogenic ability.

In the search of new drug delivery carriers for the brain, self-assembled nanoparticles (NP) were prepared from poly(N,N-dimethylacrylamide)-block-polystyrene polymer. NP displayed biocompatibility on cultured endothelial cells, macrophages and differentiated SH-SY5Y neuronal-like cells. The surface-functionalization of NP with a modified fragment of human Apolipoprotein E (mApoE) enhanced the uptake of NP by cultured human brain capillary endothelial cells, as assessed by confocal microscopy, and their permeability through a Transwell Blood Brain Barrier model made with the same cells, as assessed by fluorescence. Finally, mApoE-NP embedding doxorubicin displayed an enhanced release of drug at low pH, suggesting the potential use of these NP for the treatment of brain tumors. In the search of novel drug carriers for brain drug delivery, poly(N,N-dimethylacrylamide)-b-polystyrene nanoparticles (NP) have been functionalized with a modified fragment of Apolipoprotein E (mApoE). These NP show to be uptaken and to overcome a Blood-Brain Barrier (BBB) in vitro cellular model. The cytotoxicity of NP is also investigated on different cell lines, showing high biocompatibility.

Their physicochemical properties and relatively low cost make cellulose nanocrystals (CNCs) a potential candidate for future large-scale production in many fields including nanomedicine. Prior to a sustained and responsible development as theranostic agents, robust and reliable data concerning their safety, biocompatibility, and tissue distribution should be provided. In the present study, CNCs were extracted from Whatman filters functionalized with a fluorescent dye, and their interaction with living organisms has been thoroughly assessed. Our experimental evidence demonstrated that CNCs (1) are well tolerated by healthy mice after systemic injection; (2) are rapidly excreted, thus avoiding bioaccumulation in filter organs such as the kidneys and liver; (3) transiently migrate in bones; and (4) are able to penetrate in the cytoplasm of cancer cells without inducing material-related detrimental effects in terms of cell survival. Our results strongly suggest that the peculiar tropism to the bones is due to the chemical interaction between the Ca2+ of the bone matrix and the active surface of negatively-charged CNCs. This feature, together with the ability to penetrate cancer cells, makes CNCs a potential nanodevice for theranostics in bone tumors.

Great interest is currently being devoted to the development of nanoparticles (NPs) for biomedical purposes, designed to improve the pharmacokinetic profile of their cargos (either imaging probes or drugs) and to enhance the specific targeting at the disease site. Recent works suggest that Surface Plasmon Resonance (SPR), widely used for the analysis of biomolecular interactions, represents a technique of choice for rapid and quantitative analyses of the interaction between NPs-functionalized with specific ligands-and their putative biological targets. Moreover, SPR can provide important details on the formation and the role of the protein 'corona', i.e., the protein layer which coats NPs once they come into contact with biological fluids. These novel applications of SPR sensors may be very useful to characterize, screen and develop nanodevices for biomedical purposes. © 2012 by the authors; licensee MDPI, Basel, Switzerland.

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. From the Clinical Editor: Re and her collaborators present a method for delivering nanoliposomes via the blood brain barrier by utilizing peptide fragments including monomers or tandem dimers ApoE. This method may enable enhanced nanoliposome associated drug delivery via the blood-brain barrier, which would have enormous significance in neurodegenerative and other CNS disorders