Melissa A. Moss

Synthesis and characterization of thermally responsive pluronic F127-chitosan nanocapsules for controlled release and intracellular delivery of small molecules

In this study, we synthesized empty core-shell structured nanocapsules of Pluronic F127 and chitosan and characterized the thermal responsiveness of the nanocapsules in size and wall-permeability. Moreover, we determined the feasibility of using the nanocapsules to encapsulate small molecules for temperature-controlled release and intracellular delivery. The nanocapsules are ∼37 nm at 37 °C and expand to ∼240 nm when cooled to 4 °C in aqueous solutions, exhibiting >200 times change in volume. Moreover, the permeability of the nanocapsule wall is high at 4 °C (when the nanocapsules are swollen), allowing free diffusion of small molecules (ethidium bromide, MW = 394.3 Da) across the wall, while at 37 °C (when the nanocapsules are swollen), the wall-permeability is so low that the small molecules can be effectively withheld in the nanocapsule for hours. As a result of their thermal responsiveness in size and wall-permeability, the nanocapsules are capable of encapsulating the small molecules for temperature-controlled release and intracellular delivery into the cytosol of both cancerous (MCF-7) and noncancerous (C3H10T1/2) mammalian cells. The cancerous cells were found to take up the nanocapsules much faster than the noncancerous cells during 45 min incubation at 37 °C. Moreover, toxicity of the nanocapsules as a delivery vehicle was found to be negligible. The Pluronic F127-chitosan nanocapsules should be very useful for encapsulating small therapeutic agents to treat diseases particularly when it is combined with cryotherapy where the process of cooling and heating between 37 °C and hypothermic temperatures is naturally done.

Soluble aggregates of the amyloid‐β protein selectively stimulate permeability in human brain microvascular endothelial monolayers

Cerebral amyloid angiopathy associated with Alzheimer’s disease is characterized by cerebrovascular deposition of the amyloid‐β protein (Aβ). Aβ elicits a number of morphological and biochemical alterations in the cerebral microvasculature, which culminate in hemorrhagic stroke. Among these changes, compromise of the blood‐brain barrier has been described in Alzheimer’s disease brain, transgenic animal models of Alzheimer’s disease, and cell culture experiments. In the current study, presented data illustrates that isolated soluble Aβ1–40 aggregates, but not unaggregated monomer or mature fibril, enhance permeability in human brain microvascular endothelial monolayers. Aβ1–40‐induced changes in permeability are paralleled by both a decrease in transendothelial electrical resistance and a re‐localization of the tight junction‐associated protein zonula occludin‐1 away from cell borders and into the cytoplasm. Small soluble Aβ1–40 aggregates are confirmed to be the most potent stimulators of endothelial monolayer permeability by establishing an inverse relationship between average aggregate size and stimulated changes in diffusional permeability coefficients. These results support previous findings demonstrating that small soluble Aβ1–40 aggregates are also primarily responsible for endothelial activation, suggesting that these same species may elicit other changes in the cerebrovasculature associated with cerebral amyloid angiopathy and Alzheimer’s disease.

Unraveling the Early Events of Amyloid-β Protein (Aβ) Aggregation: Techniques for the Determination of Aβ Aggregate Size

The aggregation of proteins into insoluble amyloid fibrils coincides with the onset of numerous diseases. An array of techniques is available to study the different stages of the amyloid aggregation process. Recently, emphasis has been placed upon the analysis of oligomeric amyloid species, which have been hypothesized to play a key role in disease progression. This paper reviews techniques utilized to study aggregation of the amyloid-β protein (Aβ) associated with Alzheimers disease. In particular, the review focuses on techniques that provide information about the size or quantity of oligomeric Aβ species formed during the early stages of aggregation, including native-PAGE, SDS-PAGE, Western blotting, capillary electrophoresis, mass spectrometry, fluorescence correlation spectroscopy, light scattering, size exclusion chromatography, centrifugation, enzyme-linked immunosorbent assay, and dot blotting.

Inhibition of amyloid-β aggregation by coumarin analogs can be manipulated by functionalization of the aromatic center.

Aggregation of the amyloid-β protein (Aβ) plays a pathogenic role in the progression of Alzheimers disease, and small molecules that attenuate Aβ aggregation have been identified toward a therapeutic strategy that targets the diseases underlying cause. Compounds containing aromatic structures have been repeatedly reported as effective inhibitors of Aβ aggregation, but the functional groups that influence inhibition by these aromatic centers have been less frequently explored. The current study identifies analogs of naturally occurring coumarin as novel inhibitors of Aβ aggregation. Derivatization of the coumarin structure is shown to affect inhibitory capabilities and to influence the point at which an inhibitor intervenes within the nucleation dependent Aβ aggregation pathway. In particular, functional groups found within amyloid binding dyes, such as benzothiazole and triazole, can improve inhibition efficacy. Furthermore, inhibitor intervention at early or late stages within the amyloid aggregation pathway is shown to correlate with the ability of these functional groups to recognize and bind amyloid species that appear either early or late within the aggregation pathway. These results demonstrate that functionalization of small aromatic molecules with recognition elements can be used in the rational design of Aβ aggregation inhibitors to not only enhance inhibition but to also manipulate the inhibition mechanism.

Quartz crystal microbalance analysis of growth kinetics for aggregation intermediates of the amyloid-β protein

Evidence linking soluble aggregation intermediates of the amyloid-beta protein (A beta), as well as the ongoing growth of A beta aggregates, to physiological responses characteristic of Alzheimers disease (AD) indicates that a kinetic description A beta aggregation intermediate growth may be fundamental to understanding disease progression. Although the growth of mature A beta fibrils has been investigated using several experimental platforms, the growth of A beta aggregation intermediates has been less thoroughly explored. In the current study, a quartz crystal microbalance (QCM) was employed to analyze the real-time growth of A beta(1-40) aggregation intermediates selectively immobilized on the crystal surface. Immobilization permitted quantitative evaluation of A beta(1-40) aggregation intermediate growth under controlled solution conditions. Elongation of A beta(1-40) aggregation intermediates via monomer addition proceeded in a nonsaturable and reversible fashion. The rate of elongation was observed to vary linearly with both monomer concentration and immobilized aggregate density, to be elevated by increases in solution ionic strength, and to increase as solution pH became more acidic. Elongation was consistent with a first-order kinetic model for the single growth phase observed. These findings extend previous kinetic studies involving the growth of mature A beta fibrils to describe the growth of A beta(1-40) aggregation intermediates via monomer addition.

Soluble aggregates of the amyloid-β protein activate endothelial monolayers for adhesion and subsequent transmigration of monocyte cells

Increasing evidence suggests that the deposition of amyloid plaques, composed primarily of the amyloid‐β protein (Aβ), within the cerebrovasculature is a frequent occurrence in Alzheimer’s disease and may play a significant role in disease progression. Accordingly, the pathogenic mechanisms by which Aβ can alter vascular function may have therapeutic implications. Despite observations that Aβ elicits a number of physiological responses in endothelial cells, ranging from alteration of protein expression to cell death, the Aβ species accountable for these responses remains unexplored. In the current study, we show that isolated soluble Aβ aggregation intermediates activate human brain microvascular endothelial cells for both adhesion and subsequent transmigration of monocyte cells in the absence of endothelial cell death and monolayer disruption. In contrast, unaggregated Aβ monomer and mature Aβ fibril fail to induce any change in endothelial adhesion or transmigration. Correlations between average Aβ aggregate size and observed increases in adhesion illustrate that smaller soluble aggregates are more potent activators of endothelium. These results support previous studies demonstrating heightened neuronal activity of soluble Aβ aggregates, including Aβ‐derived diffusible ligands, oligomers, and protofibrils, and further show that soluble aggregates also selectively exhibit activity in a vascular cell model.

Soluble aggregates of the amyloid-β peptide are trapped by serum albumin to enhance amyloid-β activation of endothelial cells

BackgroundSelf-assembly of the amyloid-β peptide (Aβ) has been implicated in the pathogenesis of Alzheimers disease (AD). As a result, synthetic molecules capable of inhibiting Aβ self-assembly could serve as therapeutic agents and endogenous molecules that modulate Aβ self-assembly may influence disease progression. However, increasing evidence implicating a principal pathogenic role for small soluble Aβ aggregates warns that inhibition at intermediate stages of Aβ self-assembly may prove detrimental. Here, we explore the inhibition of Aβ1–40 self-assembly by serum albumin, the most abundant plasma protein, and the influence of this inhibition on Aβ1–40 activation of endothelial cells for monocyte adhesion.ResultsIt is demonstrated that serum albumin is capable of inhibiting in a dose-dependent manner both the formation of Aβ1–40 aggregates from monomeric peptide and the ongoing growth of Aβ1–40 fibrils. Inhibition of fibrillar Aβ1–40 aggregate growth is observed at substoichiometric concentrations, suggesting that serum albumin recognizes aggregated forms of the peptide to prevent monomer addition. Inhibition of Aβ1–40 monomer aggregation is observed down to stoichiometric ratios with partial inhibition leading to an increase in the population of small soluble aggregates. Such partial inhibition of Aβ1–40 aggregation leads to an increase in the ability of resulting aggregates to activate endothelial cells for adhesion of monocytes. In contrast, Aβ1–40 activation of endothelial cells for monocyte adhesion is reduced when more complete inhibition is observed.ConclusionThese results demonstrate that inhibitors of Aβ self-assembly have the potential to trap small soluble aggregates resulting in an elevation rather than a reduction of cellular responses. These findings provide further support that small soluble aggregates possess high levels of physiological activity and underscore the importance of resolving the effect of Aβ aggregation inhibitors on aggregate size.

Rationally designed peptoids modulate aggregation of amyloid-beta 40.

Alzheimers disease (AD) is the most common form of dementia and the sixth leading cause of death in the United States. Plaques composed of aggregated amyloid-beta protein (Aβ) accumulate between the neural cells in the brain and are associated with dementia and cellular death. Many strategies have been investigated to prevent Aβ self-assembly into disease-associated β-sheet amyloid aggregates; however, a promising therapeutic has not yet been identified. In this study, a peptoid-based mimic of the peptide KLVFF (residues 16-20 of Aβ) was tested for its ability to modulate Aβ aggregation. Peptoid JPT1 includes chiral, aromatic side chains to induce formation of a stable helical secondary structure that allows for greater interaction between the aromatic side chains and the cross β-sheet of Aβ. JPT1 was found to modulate Aβ40 aggregation, specifically decreasing lag time to β-sheet aggregate formation as well as the total number of fibrillar, β-sheet structured aggregates formed. These results suggest that peptoids may be able to limit the formation of Aβ aggregates that are associated with AD.

Impact of phospholipid bilayer saturation on amyloid-β protein aggregation intermediate growth: A quartz crystal microbalance analysis

Evidence that membrane-associated amyloid aggregate growth can impart membrane damage represents one possible mechanism for the neurodegeneration associated with deposited amyloid-beta protein (Abeta) aggregates in the brains of Alzheimers disease (AD) patients. This potential pathogenic event necessitates an understanding of the impact that cellular membrane composition may have on Abeta aggregate growth. In the current study, a quartz crystal microbalance (QCM) was employed to examine the growth of Abeta(1-40) aggregation intermediates on supported phospholipid bilayers (SPBs) assembled at the crystal surface. These surface-specific measurements illustrate that zwitterionic SPBs selectively bind aggregated but not monomeric protein, and these bound aggregates are capable of supporting nonsaturable reversible growth via monomer addition. Growth-capable Abeta(1-40) aggregation intermediates more readily bind SPBs composed of phospholipids with a greater degree of carbon saturation. Furthermore, kinetic analysis afforded by the quantitative real-time QCM measurements reveals that SPBs with greater saturation also better support the growth of bound Abeta(1-40) aggregation intermediates as a result of the slower dissociation of bound monomer rather than more efficient recognition between aggregate and monomeric protein. These findings correlate with epidemiological and experimental evidence that links increased dietary intake of polyunsaturated fatty acids to a reduced risk of AD.

Comparative Study of Inhibition at Multiple Stages of Amyloid-β Self-Assembly Provides Mechanistic Insight

The “amyloid cascade hypothesis,” linking self-assembly of the amyloid-β protein (Aβ) to the pathogenesis of Alzheimers disease, has led to the emergence of inhibition of Aβ self-assembly as a prime therapeutic strategy for this currently unpreventable and devastating disease. The complexity of Aβ self-assembly, which involves multiple reaction intermediates related by nonlinear and interconnected nucleation and growth mechanisms, provides multiple points for inhibitor intervention. Although a number of small-molecule inhibitors of Aβ self-assembly have been identified, little insight has been garnered concerning the point at which these inhibitors intervene within the Aβ assembly process. In the current study, a julolidine derivative is identified as an inhibitor of Aβ self-assembly. To gain insight into the mechanistic action of this inhibitor, the inhibition of fibril formation from monomeric protein is assessed quantitatively and compared with the inhibition of two distinct mechanisms of growth for soluble Aβ aggregation intermediates. This compound is observed to significantly inhibit soluble aggregate growth by lateral association while having little effect on soluble aggregate elongation via monomer addition. In addition, inhibition of soluble Aβ aggregate association exhibits an IC50 with a somewhat lower stoichiometric ratio than the IC50 determined for inhibition of fibril formation from monomeric Aβ. This quantitative comparison of inhibition within multiple Aβ self-assembly assays suggests that this compound binds the lateral surface of on-pathway intermediates exhibiting a range of sizes to prevent their association with other aggregates, which is required for further assembly into mature fibrils.