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The formation of block copolymer nanoparticles is commonly obtained by switching solvent quality from good solvent for both blocks to a selective solvent for one block. The effect of the selective solvent on particle dimension, shape and inner structure has been analyzed in detail, instead less attention has been focused on the role of the common solvent. Poly(ethylene oxide)-block-poly(lactic acid) (PEO-b-PLA) is widely used for the preparation of nanometric drug delivery systems and the control of the particle morphology has relevant effects on bioactivity. In this work we investigate the effect of using different common solvents (acetone, dioxane, tetrahydrofuran, dimethylformamide) on the self-assembly behavior of narrowly dispersed PEO-b-PLA copolymers, synthesized by metal free ring-opening polymerization, with large variation of molecular weight of the hydrophobic block (from 65 to 1300 lactic units). Dynamic light scattering (DLS) and cryo-electron microscopy (cryo-EM) analyses of PEO-b-PLA nanoparticles reveal that their size and shape are strongly dependent on the hydrogen bonding ability of the common solvent used in the self-assembly process. As consequence of these variations, also the cytoxicity of the nanoparticles is affected.

Alzheimer's disease affects millions of people worldwide and this figure is continuously increasing. Currently, there is no resolutive cure for this disorder, but a valid contribution could be provided by nanomedicine, utilizing multi-functionalized nanodevices as drug vehicles with additional features of specific brain targeting. Nanomedicine may represent also a practicable strategy for the pharmaceutical industry that moved from small MW pharmaceuticals to larger biologicals, such as antibodies and nucleotides, as the next generation of drugs, leading to the challenge of effective drug delivery. This review provides a survey on the nano-based strategies for Alzheimer's disease diagnosis and treatment, aiming at enhancing the passage of candidate pharmaceuticals across the BBB, and at supporting the evaluation of new therapeutic agents targeting this disease.

Background. The radio- and chemo-resistance of glioblastoma stem-like cells (GSCs), together with their innate tumor-initiating aptitude, make this cell population a crucial target for effective therapies. However, targeting GSCs is hardly difficult and complex, due to the presence of the blood-brain barrier (BBB) and the infiltrative nature of GSCs arousing their dispersion within the brain parenchyma. Methods. Liposomes (LIPs), surface-decorated with an Apolipoprotein E-modified peptide (mApoE) to enable BBB crossing, were loaded with doxorubicin (DOXO), as paradigm of cytotoxic drug triggering immunogenic cell death (ICD). Patient-derived xenografts (PDXs) obtained by GSC intracranial injection were treated with mApoE-DOXOLIPs alone or concomitantly with radiation. Results. Our results indicated that mApoE, through the engagement of the low-density lipoprotein receptor (LDLR), promotes mApoE-DOXO-LIPs transcytosis across the BBB and confers target specificity towards GSCs. Irradiation enhanced LDLR expression on both BBB and GSCs, thus further promoting LIP diffusion and specificity. When administered in combination with radiations, mApoE-DOXO-LIPs caused a significant reduction of in vivo tumor growth due to GSC apoptosis. GSC apoptosis prompted microglia/macrophage phagocytic activity, together with the activation of the antigen-presenting machinery crucially required for anti-tumor adaptive immune response. Conclusions. Our results advocate for radiotherapy and adjuvant administration of drug-loaded, mApoE-targeted nanovectors as an effective strategy to deliver cytotoxic molecules to GSCs at the surgical tumor margins, the forefront of glioblastoma (GBM) recurrence, circumventing BBB hurdles. DOXO encapsulation proved in situ immune response activation within GBM microenvironment.

Background: Multi-target drugs have gained significant recognition for the treatment of multifactorial diseases such as depression. Under a screening study of multi-potent medicinal plants with claimed antidepressant-like activity, the phenolic-rich Annona muricata aqueous extract (AE) emerged as a moderate monoamine oxidase A (hMAO-A) inhibitor and a strong hydrogen peroxide (H2O2) scavenger. Purpose: In order to protect this extract from gastrointestinal biotransformation and to improve its permeability across the blood-brain barrier (BBB), four phospholipid nanoformulations of liposomes and phytosomes functionalized with a peptide ligand promoting BBB crossing were produced. Methods: AE and nanoformulations were characterized by HPLC-DAD-ESI-MSn, HPLC-DAD, spectrophotometric, fluorescence and dynamic light scattering methods. Cytotoxicity and permeability studies were carried out using an in vitro transwell model of the BBB, composed of immortalized human microvascular endothelial cells (hCMEC/D3), and in vitro hMAO-A inhibition and H2O2 scavenging activities were performed with all samples. Results: The encapsulation/binding of AE was more efficient with phytosomes, while liposomes were more stable, displaying a slower extract release over time. In general, phytosomes were less toxic than liposomes in hCMEC/D3 cells and, when present, cholesterol improved the permeability across the cell monolayer of all tested nanoformulations. All nanoformulations conserved the antioxidant potential of AE, while phosphatidylcholine interfered with MAO-A inhibition assay. Conclusions: Overall, phytosome formulations registered the best performance in terms of binding efficiency, enzyme inhibition and scavenging activity, thus representing a promising multipotent phenolic-rich nanoshuttle for future in vivo depression treatment.

Nanoparticles may provide a viable way for neuroprotective drugs to cross the blood-brain barrier (BBB), which limits the passage of most drugs from the peripheral circulation to the brain. Heterotelechelic polymer prodrugs comprising a neuroprotective model drug (adenosine) and a maleimide functionality were synthesized by the "drug-initiated" approach and subsequent nitroxide exchange reaction. Nanoparticles were obtained by nano-precipitation and exhibited high colloidal stability with diameters in the 162-185 nm range and narrow size distributions. Nanoparticles were then covalently surface-conjugated to different proteins (albumin, alpha 2-macroglobulin and fetuin A) to test their capability of enhancing BBB translocation. Their performances in terms of endothelial permeability and cellular uptake in an in vitro BBB model were compared to that of similar nano particles with surface-adsorbed proteins, functionalized or not with the drug. It was shown that bare NPs (i.e., NPs not surface-functionalized with proteins) without the drug exhibited significant permeability and cellular uptake, which were further enhanced by NP surface functionalization with alpha 2-macroglobulin. However, the presence of the drug at the polymer chain-end prevented efficient passage of all types of NPs through the BBB model, likely due to a decrease in the hydrophobicity of the nanoparticle surface and alteration of the protein binding/coupling, respectively. These results established a new and facile synthetic approach for the surface-functionalization of polymer nanoparticles for brain delivery purposes.

Engineered nanoparticles offer the chance to improve drug transport and delivery through biological barriers, exploiting the possibility to leave the blood circulation and traverse the endothelial vascular bed, blood-brain barrier (BBB) included, to reach their target. It is known that nanoparticles gather molecules on their surface upon contact with biological fluids, forming the "protein corona", which can affect their fate and therapeutic/diagnostic performance, yet no information on the corona's evolution across the barrier has been gathered so far. Using a cellular model of the BBB and gold nanoparticles, we show that the composition of the corona undergoes dramatic quantitative and qualitative molecular modifications during passage from the "blood" to the "brain" side, while it is stable once beyond the BBB. Thus, we demonstrate that the nanoparticle corona dynamically and drastically evolves upon crossing the BBB and that its initial composition is not predictive of nanoparticle fate and performance once beyond the barrier at the target organ.

The use of exosomes for diagnostic and disease monitoring purposes is becoming particularly appealing in biomedical research because of the possibility to study directly in biological fluids some of the features related to the organs from which exosomes originate. A paradigmatic example are brain-derived exosomes that can be found in plasma and used as a direct read-out of the status of the central nervous system (CNS). Inspired by recent remarkable development of plasmonic biosensors, we have designed a surface plasmon resonance imaging (SPRi) assay that, taking advantage of the fact that exosome size perfectly fits within the surface plasmon wave depth, allows the detection of multiple exosome subpopulations of neural origin directly in blood. By use of an array of antibodies, exosomes derived from neurons and oligodendrocytes were isolated and detected with good sensitivity. Subsequently, by injecting a second antibody on the immobilized vesicles, we were able to quantify the amount of CD81 and GM1, membrane components of exosomes, on each subpopulation. In this way, we have been able to demonstrate that they are not homogeneously expressed but exhibit a variable abundance according to the exosome cellular origin. These results confirm the extreme variability of exosome composition and demonstrate how SPRi can provide an effective tool for their characterization. Besides, our work paves the road toward more precise clinical studies on the use of exosomes as potential biomarkers of neurodegenerative diseases.

Aim: To investigate if and how the ability of liposomes, previously designed for Alzheimer's therapy, to reach the brain changes in aging/pathological conditions with respect to the healthy state. Methods: Biodistribution and pharmacokinetics of liposomes in young or aged healthy mice and in an Alzheimer's mouse model were measured by radiochemical techniques. The expression of brain receptors and structural proteins was evaluated by Western blot. Results: At equal blood levels, the amount and integrity of liposomes in the brain were dramatically lower in Alzheimer's or aged mice, with respect to young animals. These differences are likely attributable to molecular alterations in the brain vasculature. Conclusion: Brain alterations in pathology or aging should be considered in the design of drug delivery systems for brain targeting

Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves

Objective: Abeta leads to neurodegeneration and memory impairment via critical signal transduction processes, both at central and peripheral level. Studying the oligomeric-Abeta toxicity could helpful to clarify these phenomena, and to develop therapeutic and effective prevention technologies. Nanoparticles (NPs) biocompatibility and their ability to modulate Abeta toxicity were investigated in primary cultures from human fibroblasts. Methods: Control fibroblasts were incubated with Abeta oligomers and NP, functionalized or not with a ApoE monomer (mApo). By MTT assay we evaluate energetic metabolism and cell viability. Western Blot and p-Elisa were performed to evidence any modulation of MAPK and Tau hyperphosphorylation. By uptake studies and confocal images, we investigated the involvement of NPs in modulating the Abeta-induced excitotoxicity and their interaction with oligomers. By commercial kit, malondialdehyde levels were tested. Results: NPs do not affect cell viability, they prevent the Abeta-induced deficit of mitochondrial activity (p<0.001) and MAPK phosphorylation (p<0.001). mApo cross cell membrane, bind oligomers and modulate glutamate uptake in a dose dependent manner (p<0.01); they counteract the increase of Abeta–induced lipid peroxidation (p<0.002) and Tau-hyperphosphorylation (p<0.0001). Conclusion: These results suggest that NPs could represent a useful tool to antagonize Abeta oligomers toxicity, counteracting the cellular stress, protein epigenetic regulation and glutamate cytotoxicity. The modulations of MAPK pathways, altered by Abeta oligomers, might clarify any molecular mechanisms that mirror those involved in the early AD phases, and NPs may be useful to study new potential therapeutic strategies.

Surface functionalization with antitransferrin receptor (TfR) mAbs has been suggested as the strategy to enhance the transfer of nanoparticles (NPs) across the blood-brain barrier (BBB) and to carry nonpermeant drugs from the blood into the brain. However, the efficiency of BBB crossing is currently too poor to be used in vivo. In the present investigation, we compared 6 different murine mAbs specific for different epitopes of the human TfR to identify the best performing one for the functionalization of NPs. For this purpose, we compared the ability of mAbs to cross an in vitro BBB model made of human brain capillary endothelial cells (hCMEC/D3). Liposomes functionalized with the best performing mAb (MYBE/4C1) were uptaken, crossed the BBB in vitro, and facilitated the BBB in vitro passage of doxorubicin, an anticancer drug, 3.9 folds more than liposomes functionalized with a nonspecific IgG, as assessed by confocal microscopy, radiochemical techniques, and fluorescence, and did not modify the cell monolayer structural or functional properties. These results show that MYBE/4C1 antihuman TfR mAb is a powerful resource for the enhancement of BBB crossing of NPs and is therefore potentially useful in the treatment of neurologic diseases and disorders including brain carcinomas.

Objectives. To understand brain ischemia injury contribution to Alzheimer’s disease (AD) onset, looking for post-translational modifications, turnover alterations and AD hallmarks secretion such as tau and amyloid precursor protein (APP), associated to autophagy activation. Methods. Rat cultured hippocampal neurons are subjected to oxygen glucose deprivation (OGD) followed by normal culture condition restoration (R), mimicking an ischemia/reperfusion (I/R) event. Afterwards AD hallmarks and autophagy markers expression and secretion are analyzed in the cells lysate and in microvescicles (MVs) isolated from culture medium. Confocal microscopy is employed to evaluate the potential re- and co-localization of tau and autophagic markers after treatment. Results. In our cellular model subjected to OGD/R, we observed tau and APP post-translational and protein level modifications in parallel to the autophagic markers (LC3II and beclin-1) expression alterations. Moreover, OGD/R treatment elicits a time-dependent increase in tau and APP C-terminal fragments (CTF) secretion by means of a heterogeneous MV population, including a group of LC3II positive vescicles. Preliminary data obtained by confocal microscopy suggest that LC3 might undergo re-localization after OGD/R treatment. Conclusions. The obtained results suggest that OGD treatment leads to multiple unconventional secretion mechanisms for tau and APP CTFs, in which autophagy activation seems to partially outcome in the exophagy secretory pathway. Although exophagy might be a defensive mechanism to prevent intracellular accumulation of misfolded proteins under conventional autophagy alteration, the secretion of misprocessed proteins has been recently associated to dendritic degeneration and neuronal death becoming a possible link to Alzheimer’s disease.

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.

The efficacy, cellular uptake and specific transport of drugs and/or imaging agents to target organs, tissues and cells are common issues in the diagnosis and treatment of different disorders. In the case of neurodegenerative diseases, they represent complex problems, since brain targeting remains a still unsolved challenge in pharmacology, due to the presence of the blood-brain barrier, a tightly packed layer of endothelial cells that prevents unwanted substances to enter the brain. Engineered nanomaterials, objects with dimensions of 1-100. nm, are providing interesting biomedical tools potentially able to solve these problems, thanks to their physico-chemical features and to the possibility of multi-functionalization, allowing to confer them different features at the same time, including the ability to cross the blood-brain barrier. This review focuses on the state-of-the-art of nanomaterials suitable for therapy and diagnostic imaging of the most common neurodegenerative disorders, as well as for neuroprotection and neuronal tissue regeneration. Finally, their potential neurotoxicity is discussed, and future nanotechnological approaches are described. © 2012 Elsevier Inc.

Figure Persented: Large-scale fabrication of graphene-based devices is an aspect of great importance for various applications including chemical and biological sensing. Toward this goal, we present here a novel chemical route for the site-specific realization of devices based on reduced graphene oxide (RGO). Electrodes patterned by photolithography are modified with amino functional groups through electrodeposition. The amine groups function as hooks for the attachment of graphene oxide flakes selectively onto the electrodes. Graphene-like electrical behavior is attained by a subsequent thermal annealing step. We show that this anchoring strategy can be scaled-up to obtain RGO devices at a wafer scale in a facile manner. The scalability of our approach coupled with the use of photolithography is promising for the rapid realization of graphene-based devices. We demonstrate one possible application of the fabricated RGO devices as electrical biosensors through the immunodetection of amyloid beta peptide. © 2012 American Chemical Society.

Alzheimer's Disease (AD) is a neurodegenerative disorder that affects millions of individuals worldwide. Accumulation of amyloid-peptide (A beta) in the brain, and its aggregation into oligomers, fibrils and plaques, plays a central role in the onset and development of AD. Starting from this observation, the E.C. FP7 project "NAD" (Nanoparticles for therapy and diagnosis of Alzheimer's disease) is involved in the design of nanoparticles that recognize and remove brain All Previous investigations by NAD Consortium have already produced nanoparticles containing anionic phospholipids or curcumin-analogues able to bind A beta with very high affinity, to inhibit fibril formation and to reduce A beta toxicity in-vitro. Starting from the observation that ganglioside GT1b binds A beta in vitro, we have synthesized liposomes, composed of sphingomyelin and cholesterol and containing GT1b ganglioside, and investigated their affinity towards A beta peptide. Surface Plasmon Resonance experiments showed a good interaction of liposomes with A beta fibrils, displaying Kd values between 125 and 150 nM. Moreover, A beta aggregation into fibrils, measured by Thioflavin T and Congo Red binding assays, was reduced of about 50%, after two weeks of monomeric peptide incubation in the presence of GT1b-containing liposomes. The ability of GT1b-containing liposomes, and the other liposomes previously described by NAD research, to bind A beta and to reduce fibrils formation, increases the interest in studying them as possible future diagnostic and therapeutic tools for the treatment of Alzheimer Disease