Fluorimetric detection of the earliest events in amyloid ß oligomerization and its inhibition by pharmacologically active liposomes.

BACKGROUND: Amyloid ß (Aß) peptide aggregation is the main molecular mechanism underlying the development of Alzheimer's disease, the most widespread form of senile dementia worldwide. Increasing evidence suggests that the key factor leading to impaired neuronal function is accumulation of water-soluble Aß oligomers rather than formation of the senile plaques created by the deposition of large fibrillary aggregates of Aß. However, several questions remain about the preliminary steps and the progression of Aß oligomerization. METHODS: We show that the initial stages of the aggregation of fluorescently labeled Aß can be determined with a high degree of precision and at physiological (i.e., nanomolar) concentrations by using either steady-state fluorimetry or time-correlated single-photon counting. RESULTS: We study the dependence of the oligomerization extent and rate on the Aß concentration. We determine the chemical binding affinity of fluorescently labeled Aß for liposomes that have been recently shown to be pharmacologically active in vivo, reducing the Aß burden within the brain. We also probe their capacity to hinder the Aß oligomerization process in vitro. CONCLUSIONS: We introduced a fluorescence assay allowing investigation of the earliest steps of Aß oligomerization, the peptide involved in Alzheimer's disease. The assay proved to be sensitive even at Aß concentrations as low as those physiologically observed in the cerebrospinal fluid. GENERAL SIGNIFICANCE: This work represents an extensive and quantitative study on the initial events of Aß oligomerization at physiological concentration. It may enhance our comprehension of the molecular mechanisms leading to Alzheimer's disease, thus paving the way to novel therapeutic strategies.

The hunt for brain Aß oligomers by peripherally circulating multi-functional nanoparticles: Potential therapeutic approach for Alzheimer disease.

We previously showed the ability of liposomes bi-functionalized with phosphatidic acid and an ApoE-derived peptide (mApoE-PA-LIP) to reduce brain Aß in transgenic Alzheimer mice. Herein we investigated the efficacy of mApoE-PA-LIP to withdraw Aß peptide in different aggregation forms from the brain, using a transwell cellular model of the blood-brain barrier and APP/PS1 mice. The spontaneous efflux of Aß oligomers (Aßo), but not of Aß fibrils, from the 'brain' side of the transwell was strongly enhanced (5-fold) in presence of mApoE-PA-LIP in the 'blood' compartment. This effect is due to a withdrawal of Aßo exerted by peripheral mApoE-PA-LIP by sink effect, because, when present in the brain side, they did not act as Aßo carrier and limit the oligomer efflux. In vivo peripheral administration of mApoE-PA-LIP significantly increased the plasma Aß level, suggesting that Aß-binding particles exploiting the sink effect can be used as a therapeutic strategy for Alzheimer disease. From the Clinical Editor: Alzheimer disease (AD) at present is an incurable disease, which is thought to be caused by an accumulation of amyloid-ß (Aß) peptides in the brain. Many strategies in combating this disease have been focused on either the prevention or dissolving these peptides. In this article, the authors showed the ability of liposomes bi-functionalized with phosphatidic acid and with an ApoE- derived peptide to withdraw amyloid peptides from the brain. The data would help the future design of more novel treatment for Alzheimer disease.

Multifunctional liposomes reduce brain ß-amyloid burden and ameliorate memory impairment in Alzheimer's disease mouse models.

Alzheimer's disease is characterized by the accumulation and deposition of plaques of ß-amyloid (Aß) peptide in the brain. Given its pivotal role, new therapies targeting Aß are in demand. We rationally designed liposomes targeting the brain and promoting the disaggregation of Aß assemblies and evaluated their efficiency in reducing the Aß burden in Alzheimer's disease mouse models. Liposomes were bifunctionalized with a peptide derived from the apolipoprotein-E receptor-binding domain for blood-brain barrier targeting and with phosphatidic acid for Aß binding. Bifunctionalized liposomes display the unique ability to hinder the formation of, and disaggregate, Aß assemblies in vitro (EM experiments). Administration of bifunctionalized liposomes to APP/presenilin 1 transgenic mice (aged 10 months) for 3 weeks (three injections per week) decreased total brain-insoluble Aß1-42 (-33%), assessed by ELISA, and the number and total area of plaques (-34%) detected histologically. Also, brain Aß oligomers were reduced (-70.5%), as assessed by SDS-PAGE. Plaque reduction was confirmed in APP23 transgenic mice (aged 15 months) either histologically or by PET imaging with [(11)C]Pittsburgh compound B (PIB). The reduction of brain Aß was associated with its increase in liver (+18%) and spleen (+20%). Notably, the novel-object recognition test showed that the treatment ameliorated mouse impaired memory. Finally, liposomes reached the brain in an intact form, as determined by confocal microscopy experiments with fluorescently labeled liposomes. These data suggest that bifunctionalized liposomes destabilize brain Aß aggregates and promote peptide removal across the blood-brain barrier and its peripheral clearance. This all-in-one multitask therapeutic device can be considered as a candidate for the treatment of Alzheimer's disease.

Liposomes bi-functionalized with phosphatidic acid and an ApoE-derived peptide affect Aß aggregation features and cross the blood-brain-barrier: implications for therapy of Alzheimer disease.

Targeting amyloid-ß peptide (Aß) within the brain is a strategy actively sought for therapy of Alzheimer's disease (AD). We investigated the ability of liposomes bi-functionalized with phosphatidic acid and with a modified ApoE-derived peptide (mApoE-PA-LIP) to affect Aß aggregation/disaggregation features and to cross in vitro and in vivo the blood-brain barrier (BBB). Surface plasmon resonance showed that bi-functionalized liposomes strongly bind Aß (kD=0.6 µM), while Thioflavin-T and SDS-PAGE/WB assays show that liposomes inhibit peptide aggregation (70% inhibition after 72 h) and trigger the disaggregation of preformed aggregates (60% decrease after 120 h incubation). Moreover, experiments with dually radiolabelled LIP suggest that bi-functionalization enhances the passage of radioactivity across the BBB either in vitro (permeability=2.5×10(-5) cm/min, 5-fold higher with respect to mono-functionalized liposomes) or in vivo in healthy mice. Taken together, our results suggest that mApoE-PA-LIP are valuable nanodevices with a potential applicability in vivo for the treatment of AD. From the clinical editor: Bi-functionalized liposomes with phosphatidic acid and a modified ApoE-derived peptide were demonstrated to influence Aß aggregation/disaggregation as a potential treatment in an Alzheimer's model. The liposomes were able to cross the blood-brain barrier in vitro and in vivo. Similar liposomes may become clinically valuable nanodevices with a potential applicability for the treatment of Alzheimer's disease.

Mesoporous silica nanoparticles trigger mitophagy in endothelial cells and perturb neuronal network activity in a size- and time-dependent manner.

PURPOSE: Mesoporous silica nanoparticles (MSNPs) are excellent candidates for biomedical applications and drug delivery to different human body areas, the brain included. Although toxicity at cellular level has been investigated, we are still far from using MSNPs in the clinic, because the mechanisms involved in the cellular responses activated by MSNPs have not yet been elucidated. MATERIALS AND METHODS: This study used an in vitro multiparametric approach to clarify relationships among size, dose, and time of exposure of MSNPs (0.05-1 mg/mL dose range), and cellular responses by analyzing the morphology, viability, and functionality of human vascular endothelial cells and neurons. RESULTS: The results showed that 24 hours of exposure of endothelial cells to 250 nm MSNPs exerted higher toxicity in terms of mitochondrial activity and membrane integrity than 30 nm MSN at the same dose. This was due to induced cell autophagy (in particular mitophagy), probably consequent to MSNP cellular uptake (>20%). Interestingly, after 24 hours of treatment with 30 nm MSNPs, very low MSNP uptake (<1%) and an increase in nitric oxide production (30%, P<0.01) were measured. This suggests that MSNPs were able to affect endothelial functionality from outside the cells. These differences could be attributed to the different protein-corona composition of the MSNPs used, as suggested by sodium dodecyl sulfate polyacrylamide-gel electrophoresis analysis of the plasma proteins covering the MSNP surface. Moreover, doses of MSNPs up to 0.25 mg/mL perturbed network activity by increasing excitability, as detected by multielectrode-array technology, without affecting neuronal cell viability. CONCLUSION: These results suggest that MSNPs may be low-risk if prepared with a diameter <30 nm and if they reach human tissues at doses <0.25 mg/mL. These important advances could help the rational design of NPs intended for biomedical uses, demonstrating that careful toxicity evaluation is necessary before using MSNPs in patients.

Investigation of Functionalized Poly(N,N-dimethylacrylamide)-block-polystyrene Nanoparticles As Novel Drug Delivery System to Overcome the Blood-Brain Barrier In Vitro.

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.