Hadas Skaat

Synthesis of fluorescent-maghemite nanoparticles as multimodal imaging agents for amyloid-β fibrils detection and removal by a magnetic field

Early diagnosis in Alzheimers disease (AD), before the onset of marked clinical symptoms, is critical in preventing the irreversible neuronal damage that eventually leads to dementia and ultimately death. Therefore, there is an urgent need for in vivo imaging agents, which are valuable as specific biomarkers to demonstrate the location and density of amyloid plaques in the living human brain. The present manuscript describes a novel method for selective marking of Abeta(40) fibrils by non-fluorescent gamma-Fe(2)O(3) and fluorescent-magnetic gamma-Fe(2)O(3)-rhodamine or gamma-Fe(2)O(3)-Congo red nanoparticles, and the complete removal of the magnetized fibrils from the aqueous continuous phase by a magnetic field. These fluorescent-maghemite nanoparticles as multimodal imaging agents have a great advantage due to the combination of the magnetic and fluorescence imaging into one nanostructured system. This hybrid system, which selectively marks Abeta(40) fibrils, might enable the early detection of plaques using both MRI and fluorescence microscopy, and therefore may be applied in in vivo AD diagnosis studies. These fluorescent-magnetic nanoparticles may also be useful as selective biomarkers to detect the location and the removal of other amyloid plaques derived from different amyloidogenic proteins that lead to neurodegenerative diseases, e.g., Parkinsons, Huntingtons, mad cow, and prion diseases.

Novel magnetic fibrin hydrogel scaffolds containing thrombin and growth factors conjugated iron oxide nanoparticles for tissue engineering

Novel tissue-engineered magnetic fibrin hydrogel scaffolds were prepared by the interaction of thrombin-conjugated iron oxide magnetic nanoparticles with fibrinogen. In addition, stabilization of basal fibroblast growth factor (bFGF) was achieved by the covalent and physical conjugation of the growth factor to the magnetic nanoparticles. Adult nasal olfactory mucosa (NOM) cells were seeded in the transparent fibrin scaffolds in the absence or presence of the free or conjugated bFGF-iron oxide nanoparticles. The conjugated bFGF enhanced significantly the growth and differentiation of the NOM cells in the fibrin scaffolds, compared to the same or even five times higher concentration of the free bFGF. In the presence of the bFGF-conjugated magnetic nanoparticles, the cultured NOM cells proliferated and formed a three-dimensional interconnected network composed mainly of tapered bipolar cells. The magnetic properties of these matrices are due to the integration of the thrombin- and bFGF-conjugated magnetic nanoparticles within the scaffolds. The magnetic properties of these scaffolds may be used in future work for various applications, such as magnetic resonance visualization of the scaffolds after implantation and reloading the scaffolds via magnetic forces with bioactive agents, eg, growth factors bound to the iron oxide magnetic nanoparticles.

Engineered Polymer Nanoparticles Containing Hydrophobic Dipeptide for Inhibition of Amyloid-β Fibrillation

Protein aggregation into amyloid fibrils is implicated in the pathogenesis of many neurodegenerative diseases. Engineered nanoparticles have emerged as a potential approach to alter the kinetics of protein fibrillation process. Yet, there are only a few reports describing the use of nanoparticles for inhibition of amyloid-β 40 (Aβ(40)) peptide aggregation, involved in Alzheimers disease (AD). In the present study, we designed new uniform biocompatible amino-acid-based polymer nanoparticles containing hydrophobic dipeptides in the polymer side chains. The dipeptide residues were designed similarly to the hydrophobic core sequence of Aβ. Poly(N-acryloyl-L-phenylalanyl-L-phenylalanine methyl ester) (polyA-FF-ME) nanoparticles of 57 ± 6 nm were synthesized by dispersion polymerization of the monomer A-FF-ME in 2-methoxy ethanol, followed by precipitation of the obtained polymer in aqueous solution. Cell viability assay confirmed that no significant cytotoxic effect of the polyA-FF-ME nanoparticles on different human cell lines, e.g., PC-12 and SH-SY5Y, was observed. A significantly slow secondary structure transition from random coil to β-sheets during Aβ(40) fibril formation was observed in the presence of these nanoparticles, resulting in significant inhibition of Aβ(40) fibrillation kinetics. However, the polyA-FF-ME analogous nanoparticles containing the L-alanyl-L-alanine (AA) dipeptide in the polymer side groups, polyA-AA-ME nanoparticles, accelerate the Aβ(40) fibrillation kinetics. The polyA-FF-ME nanoparticles and the polyA-AA-ME nanoparticles may therefore contribute to a mechanistic understanding of the fibrillation process, leading to the development of therapeutic strategies against amyloid-related diseases.

Synthesis and characterization of fluorinated magnetic core?shell nanoparticles for inhibition of insulin?amyloid fibril formation

Maghemite (gamma-Fe2O3) magnetic nanoparticles of 15.0 +/- 2.1 nm are formed by nucleation followed by controlled growth of maghemite thin films on gelatin-iron oxide nuclei. Uniform magnetic gamma-Fe2O3/poly (2,2,3,3,4,4,4-heptafluorobutyl acrylate) (gamma-Fe2O3/PHFBA) core-shell nanoparticles are prepared by emulsion polymerization of the fluorinated monomer 2,2,3,3,4,4,4-heptafluorobutyl acrylate (HFBA) in the presence of the maghemite nanoparticles. The kinetics of the insulin fibrillation process in the absence and in the presence of the gamma-Fe2O3/PHFBA core-shell nanoparticles are elucidated. A significant direct slow transition from alpha-helix to beta-sheets during insulin fibril formation is observed in the presence of the gamma-Fe2O3/PHFBA nanoparticles. This is in contradiction to our previous manuscript, which illustrated that the gamma-Fe2O3 core nanoparticles do not affect the kinetics of the formation of the insulin fibrils, and to other previous publications that describe acceleration of the fibrillation process by using various types of nanoparticles. These core-shell nanoparticles may therefore be also useful for the inhibition of conformational changes of other amyloidogenic proteins that lead to neurodegenerative diseases such as Alzheimers, Parkinsons, Huntingtons, mad cow and prion diseases.

Effect of maghemite nanoparticles on insulin amyloid fibril formation: Selective labeling, kinetics, and fibril removal by a magnetic field

Maghemite (gamma-Fe(2)O(3)) magnetic nanoparticles of 15.0 +/- 2.1 nm were formed by nucleation followed by controlled growth of maghemite thin films on gelatin-iron oxide nuclei. Human insulin amyloid fibrils were formed by incubating the monomeric insulin dissolved in aqueous continuous phase at pH 1.6 and 65 degrees C. Magnetic human insulin amyloid fibrils/gamma-Fe(2)O(3) nanoparticle assemblies were prepared by interacting the gamma-Fe(2)O(3) nanoparticles with the insulin amyloid fibrils during or after their formation. The nanoparticles attached selectively to the insulin fibrils in both cases. The kinetics of the insulin fibrillation process in the absence and the presence of the gamma-Fe(2)O(3) nanoparticles was elucidated. The insulin amyloid fibrils/gamma-Fe(2)O(3) nanoparticle assemblies were easily extracted from the aqueous phase via a magnetic field. We hypothesize that this selective extraction approach may also be applicable for the removal of other amyloidogenic proteins that lead to neurodegenerative diseases (e.g., Alzheimers, Parkinsons, Huntingtons, mad cow, and prion diseases) from their continuous phase, e.g. milk, blood, neurological fluid, etc.

The Role of Neurotrophic Factors Conjugated to Iron Oxide Nanoparticles in Peripheral Nerve Regeneration: In Vitro Studies

Local delivery of neurotrophic factors is a pillar of neural repair strategies in the peripheral nervous system. The main disadvantage of the free growth factors is their short half-life of few minutes. In order to prolong their activity, we have conjugated to iron oxide nanoparticles three neurotrophic factors: nerve growth factor (βNGF), glial cell-derived neurotrophic factor (GDNF), and basic fibroblast growth factor (FGF-2). Comparative stability studies of free versus conjugated factors revealed that the conjugated neurotrophic factors were significantly more stable in tissue cultures and in medium at 37°C. The biological effects of free versus conjugated neurotrophic factors were examined on organotypic dorsal root ganglion (DRG) cultures performed in NVR-Gel, composed mainly of hyaluronic acid and laminin. Results revealed that the conjugated neurotrophic factors enhanced early nerve fiber sprouting compared to the corresponding free factors. The most meaningful result was that conjugated-GDNF, accelerated the onset and progression of myelin significantly earlier than the free GDNF and the other free and conjugated factors. This is probably due to the beneficial and long-acting effect that the stabilized conjugated-GDNF had on neurons and Schwann cells. These conclusive results make NVR-Gel enriched with conjugated-GDNF, a desirable scaffold for the reconstruction of severed peripheral nerve.

Magnetic Scaffolds Enriched with Bioactive Nanoparticles for Tissue Engineering

Novel magnetic fibrin hydrogel scaffolds for cell implantation and tissue engineering are reported. The magnetic scaffolds are produced by the interaction between thrombin-conjugated maghemite nanoparticles of narrow size distribution and fibrinogen. These scaffolds, enriched with growth factor conjugated fluorescent maghemite nanoparticles, provide a supporting 3D environment for massive proliferation of various cell types, and can be successfully visualized by MRI.

Antibody-conjugated, dual-modal, near-infrared fluorescent iron oxide nanoparticles for antiamyloidgenic activity and specific detection of amyloid-β fibrils

Amyloid-β (Aβ) peptide is the main fibrillar component of plaque deposits found in brains affected by Alzheimer’s disease (AD) and is related to the pathogenesis of AD. Passive anti-Aβ immunotherapy has emerged as a promising approach for the therapy of AD, based on the administration of specific anti-Aβ monoclonal antibodies (aAβmAbs) to delay Aβ aggregation in the brain. However, the main disadvantage of this approach is the required readministration of the aAβmAbs at frequent intervals. There are only a few reports describing in vitro study for the immobilization of aAβmAbs to nanoparticles as potential targeting agents of Aβ aggregates. In this article, we report the immobilization of the aAβmAb clone BAM10 to near-infrared fluorescent maghemite nanoparticles for the inhibition of Aβ40 fibrillation kinetics and the specific detection of Aβ40 fibrils. The BAM10-conjugated iron oxide nanoparticles were well-characterized, including their immunogold labeling and cytotoxic effect on PC-12 (pheochromocytoma cell line). Indeed, these antibody-conjugated nanoparticles significantly inhibit the Aβ40 fibrillation kinetics compared with the same concentration, or even five times higher, of the free BAM10. This inhibitory effect was confirmed by different assays such as the photo-induced crosslinking of unmodified proteins combined with sodium dodecyl sulfate– polyacrylamide gel electrophoresis. A cell viability assay also confirmed that these antibody-conjugated nanoparticles significantly reduced the Aβ40-induced cytotoxicity to PC-12 cells. Furthermore, the selective labeling of the Aβ40 fibrils with the BAM10-conjugated near-infrared fluorescent iron oxide nanoparticles enabled specific detection of Aβ40 fibrils ex vivo by both magnetic resonance imaging and fluorescence imaging. This study highlights the immobilization of the aAβmAb to dual-modal nanoparticles as a potential approach for aAβmAb delivery, eliminating the issue of readministration, and contributes to the development of multifunctional agents for diagnosis and therapy of AD.

Enhancement of the growth and differentiation of nasal olfactory mucosa cells by the conjugation of growth factors to functional nanoparticles.

Growth factors are critical components in the tissue engineering approach. Basic fibroblast growth factor (bFGF), a representative growth factor, stimulates the cellular functions of various cells and has been used extensively for the repair and regeneration of tissues. The in vivo half-life time of free bFGF is short, about 3-10 min, due to rapid enzymatic degradation. Stabilization of the bFGF was accomplished by the covalent or physical conjugation of this factor to fluorescent maghemite (γ-Fe(2)O(3)) nanoparticles. In the present study, nasal olfactory mucosa (NOM) cells from adult rats were cultured in suspension on chitosan microcarriers (MCs) in the presence of the nonconjugated or bFGF-conjugated nanoparticles, or the free factor. The floating cells/nonconjugated, conjugated, or free bFGF/MCs aggregates were then seeded in a viscous gel. In this manuscript, we are the first to report that the stabilization of the factor by its conjugation to these nanoparticles significantly improved NOM cell-proliferation properties (migration, growth, and differentiation), compared to the same concentration, or even five times higher, of the free factor. This novel approach may significantly contribute to the advancement of the tissue engineering field.

Age-dependent effects of microglial inhibition in vivo on Alzheimer’s disease neuropathology using bioactive-conjugated iron oxide nanoparticles

BackgroundTau dysfunction is believed to be the primary cause of neurodegenerative disorders referred to as tauopathies, including Alzheimer’s disease, Pick’s disease, frontotemporal dementia and Parkinsonism. The role of microglial cells in the pathogenesis of tauopathies is still unclear. The activation of microglial cells has been correlated with neuroprotective effects through the release of neurotrophic factors and through clearance of cell debris and phagocytosis of cells with intracellular inclusions. In contrast, microglial activation has also been linked with chronic neuroinflammation contributing to the development of neurodegenerative diseases such as tauopathies. Microglial activation has been recently reported to precede tangle formation and the attenuation of tau pathology occurs after immunosuppression of transgenic mice.MethodsHere we report the specific inhibition of microglial cells in rTg4510 tau-mutant mice by using fibrin γ377-395 peptide conjugated to iron oxide (γ-Fe2O3) nanoparticles of 21 ± 3.5 nm diameter.ResultsStabilization of the peptide by its covalent conjugation to the γ-Fe2O3 nanoparticles significantly decreased the number of the microglial cells compared to the same concentration of the free peptide. The specific microglial inhibition induces different effects on tau pathology in an age dependent manner. The reduction of activation of microglial cells at an early age increases the number of neurons with hyperphosphorylated tau in transgenic mice. In contrast, reduction of activation of microglial cells reduced the severity of the tau pathology in older mice. The number of neurons with hyperphosphorylated tau and the number of neurons with tangles are reduced than those in animals not receiving the fibrin γ377-395 peptide-nanoparticle conjugate.ConclusionsThese results demonstrate a differential effect of microglial activity on tau pathology using the fibrin γ377-395 peptide-nanoparticle conjugate, depending on age and/or stage of the neuropathological accumulation and aggregation.