RESUMEN
Aß peptides are known to bind neural plasma membranes in a process leading to the deposit of Aß-enriched plaques. These extracellular structures are characteristic of Alzheimer's disease, the major cause of late-age dementia. The mechanisms of Aß plaque formation and deposition are far from being understood. A vast number of studies in the literature describe the efforts to analyze those mechanisms using a variety of tools. The present review focuses on biophysical studies mostly carried out with model membranes or with computational tools. This review starts by describing basic physical aspects of lipid phases and commonly used model membranes (monolayers and bilayers). This is followed by a discussion of the biophysical techniques applied to these systems, mainly but not exclusively Langmuir monolayers, isothermal calorimetry, density-gradient ultracentrifugation, and molecular dynamics. The Methodological Section is followed by the core of the review, which includes a summary of important results obtained with each technique. The last section is devoted to an overall reflection and an effort to understand Aß-bilayer binding. Concepts such as Aß peptide membrane binding, adsorption, and insertion are defined and differentiated. The roles of membrane lipid order, nanodomain formation, and electrostatic forces in Aß-membrane interaction are separately identified and discussed.
Asunto(s)
Péptidos beta-Amiloides , Membrana Dobles de Lípidos , Lípidos de la Membrana , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/química , Humanos , Membrana Dobles de Lípidos/metabolismo , Membrana Dobles de Lípidos/química , Lípidos de la Membrana/metabolismo , Lípidos de la Membrana/química , Unión Proteica , Membrana Celular/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Fenómenos Biofísicos , Simulación de Dinámica MolecularRESUMEN
The amyloidogenic Aß peptides are widely considered as a pathogenic agent in Alzheimer's disease. Aß(1-42) would form aggregates of amyloid fibrils on the neuron plasma membranes, thus perturbing neuronal functionality. Conflicting data are available on the influence of bilayer order on Aß(1-42) binding to membranes. In the present study, a biophysical approach was used in which isothermal calorimetry and surface pressure measurements were applied to explore the interaction of Aß(1-42) in either monomeric, oligomeric, or fibrillar form with model membranes (bilayers or monolayers) in the liquid-ordered state that were either electrically neutral or negatively charged. In the latter case, this contained phosphatidic acid, cardiolipin, or ganglioside. The calorimetric studies showed that Aß(1-42) fibrils, oligomers, and monomers could bind and/or be inserted into bilayers, irrespective of electric charge, in the liquid-ordered state, except that monomers could not interact with electrically neutral bilayers. The monolayer studies in the Langmuir balance demonstrated that Aß(1-42) aggregation hindered peptide insertion into the monolayer, hindered insertion in the decreasing order of monomer > oligomer > fibril, and that lipid composition did not cause large differences in insertion, apart from a slight facilitation of monomer and oligomer insertion by gangliosides.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Humanos , Péptidos beta-Amiloides/metabolismo , Amiloide/química , Fragmentos de Péptidos/metabolismo , Enfermedad de Alzheimer/metabolismo , GangliósidosRESUMEN
AIM: The effect of liposomes bi-functionalized with phosphatidic acid and with a synthetic peptide derived from human apolipoprotein E has been evaluated on the aggregation features of different amyloidogenic proteins: human Amyloid ß1-40 (Aß1-40), transthyretin (TTR) variant S52P, human ß2microglobulin (ß2m) variants ΔN6 and D76N, Serum Amyloid A (SAA). METHODS: The formation of fibrillar aggregates of the proteins was investigated by ThioflavinT fluorescence assay and validated by Atomic Force Microscopy. RESULTS: The results show that liposomes are preventing the transition of non-aggregated forms to the fibrillar state, with stronger effects on Aß1-40, ß2m ΔN6 and SAA. Liposomes also induce disaggregation of the amyloid aggregates of all the proteins investigated, with stronger effects on Aß1-40, ß2 D76N and TTR.SPR assays show that liposomes bind Aß1-40 and SAA aggregates with high affinity (KD in the nanomolar range) whereas binding to TTR aggregates showed a lower affinity (KD in the micromolar range). Aggregates of ß2m variants showed both high and low affinity binding sites. Computed Structural analysis of protein fibrillar aggregates and considerations on the multidentate features of liposomes allow to speculate a common mechanism of action, based on binding the ß-stranded peptide regions responsible for the amyloid formation. CONCLUSION: Thus, multifunctional liposomes perform as pharmacological chaperones with anti-amyloidogenic activity, with a promising potential for the treatment of a number of protein-misfolding diseases.Key messageAmyloidosis is a group of diseases, each due to a specific protein misfolding.Anti-amyloidogenic nanoparticles have been gaining the utmost importance as a potential treatment for protein misfolding disorders.Liposomes bi-functionalized with phosphatidic acid and with a synthetic peptide derived from human apolipoprotein E showed anti-amyloidogenic activity.
Asunto(s)
Amiloide , Liposomas , Humanos , Amiloide/química , Amiloide/metabolismo , Agregado de Proteínas , Chaperonas Moleculares , Ácidos Fosfatidicos , ApolipoproteínasRESUMEN
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-DOXO-LIPs 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.
RESUMEN
Aß42 peptide binds neuronal membranes and aggregates into plaques that are characteristic of Alzheimer's disease. Aß42 peptide has been proposed to be generated in membrane (nano) domains in the liquid-ordered phase, ganglioside GM1 being a major facilitator of peptide binding to the membrane. The peptide exists in solution in various degrees of aggregation, either monomers, oligomers or fibrils, of which oligomers appear to be particularly toxic. The present study reports on the binding of Aß42 peptide, in monomer, oligomer or fibril form, to model membranes (lipid vesicles or monolayers), composed of sphingomyelin and cholesterol in equimolar ratios, to which 1-5 mol% of different gangliosides were incorporated. Thermodynamic binding parameters obtained from calorimetric data indicate a strong tendency to bind the membrane (ΔG ≈ 7 kcal/mol peptide), in a process dominated in most cases by the increase in entropy. ΔG was virtually invariant with the ganglioside species and the aggregation state of the peptide. The Langmuir balance demonstrated the capacity of all peptide preparations to become inserted in lipid monolayers of any composition and initial π in the range 10-30 mN/m, although fibrils were less capable to do so than oligomers or monomers, their maximum initial π being ≈25 mN/m.
Asunto(s)
Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Calorimetría , Colesterol/química , Gangliósido G(M1)/química , Humanos , Agregado de Proteínas , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Esfingomielinas/química , TermodinámicaRESUMEN
ß-Amyloid (Aß) is a 39-43 residue peptide involved in the pathogenesis of Alzheimer's disease. Aß deposits onto the cells and gives rise to the plaques that are characteristic of the disease. In an effort to understand the molecular mechanism of plaque formation, we have examined the interaction of Aß42, considered to be the most pathogenic of the peptides, with lipid bilayers consisting of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) to which small amounts of GM1 ganglioside (1-5 mol%) were incorporated. POPC bilayers exist in the fluid, or liquid-disordered state at room temperature, mimicking the fluidity of cell membranes. An Aß42 preparation consisting essentially of peptide monomers was used. A combination of molecular dynamics (MD), isothermal calorimetry and Langmuir balance measurements was applied. Our results show that Aß binds POPC bilayers, and that binding increases (ΔG of binding decreases) with GM1, but only up to 3 mol% of the ganglioside, larger concentrations appearing to have a lower effect. MD and Langmuir balance measurements concur in showing that the peptide adsorbs onto the bilayer surface, but does not become inserted into it at surface pressures compatible with the cell membrane conditions. Thioflavin T measurements agree with MD in revealing a very low degree of peptide oligomerization/aggregation under our conditions. This is in contrast with previous studies showing peptide aggregation and insertion when interacting with membranes in the liquid-ordered state. The present contribution underlines the importance of bilayer lipid composition and properties for Aß plaque formation.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Gangliósido G(M1)/química , Fragmentos de Péptidos/metabolismo , Fosfatidilcolinas/química , Adsorción , Péptidos beta-Amiloides/análisis , Péptidos beta-Amiloides/química , Benzotiazoles , Calorimetría , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/químicaRESUMEN
The binding of Aß42 peptide monomers to sphingomyelin/cholesterol (1:1 mol ratio) bilayers containing 5 mol% gangliosides (either GM1, or GT1b, or a mixture of brain gangliosides) has been assayed by density gradient ultracentrifugation. This procedure provides a direct method for measuring vesicle-bound peptides after non-bound fraction separation. This centrifugation technique has rarely been used in this context previously. The results show that gangliosides increase by about two-fold the amount of Aß42 bound to sphingomyelin/cholesterol vesicles. Complementary studies of the same systems using thioflavin T fluorescence, Langmuir monolayers or infrared spectroscopy confirm the ganglioside-dependent increased binding. Furthermore these studies reveal that gangliosides facilitate the aggregation of Aß42 giving rise to more extended ß-sheets. Thus, gangliosides have both a quantitative and a qualitative effect on the binding of Aß42 to sphingomyelin/cholesterol bilayers.
Asunto(s)
Péptidos beta-Amiloides/química , Colesterol/química , Gangliósidos/química , Fragmentos de Péptidos/química , Esfingomielinas/química , Fenómenos Biofísicos , Centrifugación por Gradiente de Densidad , Humanos , Membrana Dobles de Lípidos/química , Liposomas/química , Unión ProteicaRESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer , Nanopartículas , Enfermedad de Alzheimer/tratamiento farmacológico , Barrera Hematoencefálica , Sistemas de Liberación de Medicamentos , Humanos , NanomedicinaRESUMEN
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 nanoprecipitation and exhibited high colloidal stability with diameters in the 162-185â¯nm range and narrow size distributions. Nanoparticles were then covalently surface-conjugated to different proteins (albumin, α2-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 nanoparticles 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 α2-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 adecrease 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.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Nanopartículas/metabolismo , Polímeros/metabolismo , Profármacos/metabolismo , Proteínas/metabolismo , Adsorción/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Encéfalo/metabolismo , Línea Celular , Portadores de Fármacos/metabolismo , Humanos , Permeabilidad/efectos de los fármacosRESUMEN
PURPOSE: Nanotechnologies turned out to be promising in the development of diagnostic and therapeutic approaches toward neurodegenerative disorders. However, only a very scant number of nanodevices until now proved to be effective on preclinical animal models. Although specific tests in vivo are available to assess the potential toxicity of these nanodevices on cognitive functions, those to evaluate their biosafety in vitro on neurons are still to be improved. MATERIALS AND METHODS: We utilized the patch-clamp technique on primary cultures of cortical neural cells isolated from neonatal rats, aiming to evaluate their electrical properties after the incubation with liposomes (mApoE-PA-LIPs), previously proved able to cross the blood-brain barrier and to be effective on mouse models of Alzheimer's disease (AD), both in the absence and in the presence of ß-amyloid peptide oligomers. RESULTS: Data show a high degree of biocompatibility, evaluated by lactate dehydrogenase (LDH) release and MTT assay, and the lack of cellular internalization. After the incubation with mApoE-PA-LIPs, neuronal membranes show an increase in the input resistance (from 724.14±76 MΩ in untreated population to 886.06±86 MΩ in the treated one), a reduction in the rheobase current (from 29.6±3 to 24.2±3 pA in untreated and treated, respectively), and an increase of the firing frequency, consistent with an ultimate increase in intrinsic excitability. Data obtained after co-incubation of mApoE-PA-LIPs with ß-amyloid peptide oligomers suggest a retention of liposome efficacy. CONCLUSION: These data suggest the ability of liposomes to modulate neuronal electrical properties and are compatible with the previously demonstrated amelioration of cognitive functions induced by treatment of AD mice with liposomes. We conclude that this electrophysiological approach could represent a useful tool for nanomedicine to evaluate the effect of nanoparticles on intrinsic neuronal excitability.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Neuronas/metabolismo , Potenciales de Acción , Péptidos beta-Amiloides/metabolismo , Animales , Animales Recién Nacidos , Apolipoproteínas E/metabolismo , Materiales Biocompatibles/química , Supervivencia Celular , Células Cultivadas , Endocitosis , Liposomas , Masculino , Ratones , Nanopartículas/química , Ácidos Fosfatidicos/química , RatasRESUMEN
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.
Asunto(s)
Encéfalo/citología , Exosomas/química , Neuronas/química , Oligodendroglía/química , Plasma/química , Resonancia por Plasmón de Superficie/métodos , Adulto , Anticuerpos Inmovilizados/química , Femenino , Gangliósido G(M1)/análisis , Humanos , Masculino , Tetraspanina 28/análisisRESUMEN
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.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Nanopartículas/metabolismo , Corona de Proteínas/metabolismo , Barrera Hematoencefálica/química , Células Endoteliales/química , Células Endoteliales/metabolismo , Humanos , Nanopartículas/química , Corona de Proteínas/químicaRESUMEN
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.
Asunto(s)
Annona/química , Antioxidantes/farmacología , Portadores de Fármacos/química , Liposomas/química , Extractos Vegetales/administración & dosificación , Barrera Hematoencefálica/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Portadores de Fármacos/administración & dosificación , Células Endoteliales/efectos de los fármacos , Depuradores de Radicales Libres/farmacología , Humanos , Peróxido de Hidrógeno/metabolismo , Liposomas/administración & dosificación , Inhibidores de la Monoaminooxidasa/farmacología , Nanoestructuras/administración & dosificación , Nanoestructuras/química , Fenoles/farmacología , Extractos Vegetales/farmacología , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
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.
Asunto(s)
Envejecimiento/patología , Enfermedad de Alzheimer/tratamiento farmacológico , Encéfalo/efectos de los fármacos , Liposomas/administración & dosificación , Envejecimiento/efectos de los fármacos , Enfermedad de Alzheimer/patología , Animales , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/patología , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Humanos , Liposomas/química , Liposomas/farmacocinética , Ratones , Distribución TisularRESUMEN
Alzheimer's disease (AD) is a neurodegenerative disorder related, in part, to the accumulation of amyloid-ß peptide (Aß) and especially the Aß peptide 1-42 (Aß1-42). The aim of this study was to design nanocarriers able to: (i) interact with the Aß1-42 in the blood and promote its elimination through the "sink effect" and (ii) correct the memory defect observed in AD-like transgenic mice. To do so, biodegradable, PEGylated nanoparticles were surface-functionalized with an antibody directed against Aß1-42. Treatment of AD-like transgenic mice with anti-Aß1-42-functionalized nanoparticles led to: (i) complete correction of the memory defect; (ii) significant reduction of the Aß soluble peptide and its oligomer level in the brain and (iii) significant increase of the Aß levels in plasma. This study represents the first example of Aß1-42 monoclonal antibody-decorated nanoparticle-based therapy against AD leading to complete correction of the memory defect in an experimental model of AD.
Asunto(s)
Enfermedad de Alzheimer/complicaciones , Péptidos beta-Amiloides/inmunología , Anticuerpos Monoclonales/química , Modelos Animales de Enfermedad , Trastornos de la Memoria/terapia , Nanopartículas/administración & dosificación , Polímeros/administración & dosificación , Animales , Anticuerpos Monoclonales/inmunología , Humanos , Masculino , Ratones , Ratones Transgénicos , Nanopartículas/química , Nanopartículas/metabolismo , Polímeros/química , Polímeros/metabolismo , Recuperación de la FunciónRESUMEN
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.
Asunto(s)
Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/metabolismo , Espectrometría Raman , Biomarcadores , Vesículas Extracelulares/ultraestructura , Humanos , Células Madre Mesenquimatosas/ultraestructura , Tetraspanina 29/metabolismo , Tetraspanina 30/metabolismoRESUMEN
In this study, we evaluated the anti-amyloid effect of functionalized nanoliposomes (mApoE-PA-LIP) in a mouse model of Alzheimer's disease with use of positron emission tomography and ß-amyloid (Aß)-targeted tracer [11C]Pittsburgh compound B ([11C]PIB). APP23 mice were injected with mApoE-PA-LIP or saline (3 times per week for 3 weeks) and [11C]PIB imaging was performed at baseline, after the treatment and after 3 months follow-up period, accompanied by Aß immunohistochemistry and ELISA. After the treatment, [11C]PIB binding ratios between mApoE-PA-LIP and saline groups were equivalent in all analyzed brain regions; however, in the saline group, binding ratios increased from the baseline, whereas no increase was detected in the mApoE-PA-LIP group. During the additional follow-up, [11C]PIB binding increased significantly from baseline in both groups, and binding ratios correlated with the immunohistochemically defined Aß load. This study further supports the use of [11C]PIB positron emission tomography imaging as a biomarker of Aß deposition in APP23 mice and highlights the benefits of noninvasive follow-up, that is, using baseline data for animal stratification and normalization of treatment effects to baseline values, for future anti-amyloid treatment studies.
Asunto(s)
Enfermedad de Alzheimer/diagnóstico por imagen , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/metabolismo , Compuestos de Anilina , Liposomas/administración & dosificación , Liposomas/uso terapéutico , Tomografía de Emisión de Positrones , Tiazoles , Enfermedad de Alzheimer/metabolismo , Animales , Radioisótopos de Carbono , Modelos Animales de Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Liposomas/farmacología , Masculino , Ratones Transgénicos , Nanopartículas , RadiofármacosRESUMEN
The failure of clinical trials largely focused on mild to moderate stages of Alzheimer disease has suggested to the scientific community that the effectiveness of Amyloid-ß (Aß)-centered treatments should be evaluated starting as early as possible, well before irreversible brain damage has occurred. Accordingly, also the preclinical development of new therapies should be carried out taking into account this suggestion. In the present investigation we evaluated the efficacy of a treatment with liposomes multifunctionalized for crossing the blood-brain barrier and targeting Aß, carried out on young APP/PS1 Tg mice, taken as a model of pre-symptomatic disease stage. Liposomes were administered once a week to Tg mice for 7months, starting at the age of 5months and up to the age of 12 when they display AD-like cognitive and brain biochemical/anatomical features. The treatment prevented the onset of the long-term memory impairment and slowed down the deposition of brain Aß; at anatomical level, prevented both ventricle enlargement and entorhinal cortex thickness reduction, otherwise occurring in untreated mice. Strikingly, these effects were maintained 3months after treatment discontinuation. An increase of Aß levels in the liver was detected at the end of the treatment, then followed also by reduction of brain Amyloid Precursor Protein and increase of Aß-degrading enzymes. These results suggest that the treatment promotes brain Aß clearance by a peripheral 'sink' effect and ultimately affects Aß turnover in the brain. Worth of note, the treatment was apparently not toxic for all the organs analyzed, in particular for brain, as suggested by the lower brain TNF-α and MDA levels, and by higher level of SOD activity in treated mice. Together, these findings promote a very early treatment with multi-functional liposomes as a well-tolerated nanomedicine-based approach, potentially suitable for a disease-modifying therapy of AD, able to delay or prevent relevant features of the disease.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Apolipoproteínas E/uso terapéutico , Encéfalo/efectos de los fármacos , Liposomas/uso terapéutico , Trastornos de la Memoria/prevención & control , Ácidos Fosfatidicos/uso terapéutico , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Animales , Apolipoproteínas E/administración & dosificación , Apolipoproteínas E/química , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Sistemas de Liberación de Medicamentos , Liposomas/administración & dosificación , Liposomas/química , Masculino , Trastornos de la Memoria/complicaciones , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/patología , Ratones , Ratones Transgénicos , Ácidos Fosfatidicos/administración & dosificación , Ácidos Fosfatidicos/químicaRESUMEN
The accumulation of extracellular amyloid beta (Abeta42) both in brain and in cerebral vessels characterizes Alzheimer's disease (AD) pathogenesis. Recently, the possibility to functionalize nanoparticles (NPs) surface with Abeta42 binding molecules, making them suitable tools for reducing Abeta42 burden has been shown effective in models of AD. Aim of this work consisted in proving that NPs might be effective in sequestering Abeta42 in biological fluids, such as CSF and plasma. This demonstration is extremely important considering that these Abeta42 pools are in continuum with the brain parenchyma with drainage of Abeta from interstitial brain tissue to blood vessel and plasma. In this work, liposomes (LIP) were functionalized as previously shown in order to promote high-affinity Abeta binding, i.e., either with, phosphatidic acid (PA), or a modified Apolipoprotein E-derived peptide (mApo), or with a curcumin derivative (TREG); Abeta42 levels were determined by ELISA in CSF and plasma samples. mApo-PA-LIP (25 and 250 µM) mildly albeit significantly sequestered Abeta42 proteins in CSF samples obtained from healthy subjects (p < 0.01). Analogously a significant binding (â¼20%) of Abeta42 (p < 0.001) was demonstrated following exposure to all functionalized liposomes in plasma samples obtained from selected AD or Down's syndrome patients expressing high levels of Abeta42. The same results were obtained by quantifying Abeta42 content after removal of liposome-bound Abeta by using gel filtration chromatography or ultracentrifugation on a discontinuous sucrose density gradient. In conclusion, we demonstrate that functionalized liposomes significantly sequester Abeta42 in human biological fluids. These data may be critical for future in vivo administration tests using NPs for promoting sink effect.
Asunto(s)
Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/líquido cefalorraquídeo , Péptidos beta-Amiloides/sangre , Péptidos beta-Amiloides/líquido cefalorraquídeo , Liposomas/metabolismo , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/líquido cefalorraquídeo , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Femenino , Humanos , MasculinoRESUMEN
Aggregation of amyloid-ß peptide (Aß) is a key event in the pathogenesis of Alzheimer's disease (AD). We investigated the effects of nanoliposomes decorated with the retro-inverso peptide RI-OR2-TAT (Ac-rGffvlkGrrrrqrrkkrGy-NH2) on the aggregation and toxicity of Aß. Remarkably low concentrations of these peptide inhibitor nanoparticles (PINPs) were required to inhibit the formation of Aß oligomers and fibrils in vitro, with 50% inhibition occurring at a molar ratio of ~1:2000 of liposome-bound RI-OR2-TAT to Aß. PINPs also bound to Aß with high affinity (Kd=13.2-50 nM), rescued SHSY-5Y cells from the toxic effect of pre-aggregated Aß, crossed an in vitro blood-brain barrier model (hCMEC/D3 cell monolayer), entered the brains of C57 BL/6 mice, and protected against memory loss in APPSWE transgenic mice in a novel object recognition test. As the most potent aggregation inhibitor that we have tested so far, we propose to develop PINPs as a potential disease-modifying treatment for AD.