Fluorescent nanodiamonds engage innate immune effector cells: A potential vehicle for targeted anti-tumor immunotherapy.

Fluorescent nanodiamonds (FNDs) are nontoxic, infinitely photostable, and emit fluorescence in the near infrared region. Natural killer (NK) cells and monocytes are part of the innate immune system and are crucial to the control of carcinogenesis. FND-mediated stimulation of these cells may serve as a strategy to enhance anti-tumor activity. FNDs were fabricated with a diameter of 70±28 nm. Innate immune cell FND uptake, viability, surface marker expression, and cytokine production were evaluated in vitro. Evaluation of fluorescence emission from the FNDs was conducted in an animal model. In vitro results demonstrated that treatment of immune cells with FNDs resulted in significant dose-dependent FND uptake, no compromise in cell viability, and immune cell activation. FNDs were visualized in an animal model. Hence, FNDs may serve as novel agents with "track and trace" capabilities to stimulate innate immune cell anti-tumor responses, especially as FNDs are amenable to surface-conjugation with immunomodulatory molecules.

Antifungal activity against Candida albicans of starch Pickering emulsion with thymol or amphotericin B in suspension and calcium alginate films.

Conventional antifungal treatments against Candida albicans in the oral cavity often result in increased cytotoxicity. The goal of this study was to determine the potential of starch Pickering emulsion as a delivery vehicle for an antifungal natural phenolic compound such as thymol in simulated saliva fluid (SSF) compared to amphotericin B. An oil-in-water (o/w) emulsion was stabilized using starch particles. Physical stability of the emulsion and disruption induced by α-amylase activity in SSF was evaluated. Encapsulated thymol in o/w emulsion was compared to encapsulated amphotericin B for antifungal activity against C. albicans in suspension using emulsions or zone inhibition assay on agar plates using emulsions dispersed in alginate films. Results showed that the emulsions were stable for at least three weeks. Digestion of the emulsion by α-amylase led to coalescence of emulsion droplets. The antifungal activity of thymol and amphotericin B in emulsion formulation was enhanced upon incubation with α-amylase. Results from the zone inhibition assay demonstrated efficacy of the emulsions dispersed in alginate films. Interestingly, addition of α-amylase to the alginate films resulted in a decreased inhibitory effect. Overall, this study showed that starch Pickering emulsions have a potential to deliver hydrophobic antifungal compounds to treat oral candidiasis.

Rapid detection of bacteriophages in starter culture using water-in-oil-in-water emulsion microdroplets.

Bacteriophage contamination of starter culture and raw material poses a major problem in the fermentation industry. In this study, a rapid detection of lytic phage contamination in starter culture using water-in-oil-in-water (W/O/W) emulsion microdroplets was described. A model bacteria with varying concentrations of lytic phages were encapsulated in W/O/W emulsion microdroplets using a simple needle-in-tube setup. The detection of lytic phage contamination was accomplished in 1 h using the propidium iodide labeling of the phage-infected bacteria inside the W/O/W emulsion microdroplets. Using this approach, a detection limit of 10(2) PFU/mL of phages was achieved quantitatively, while 10(4) PFU/mL of phages could be detected qualitatively based on visual comparison of the fluorescence images. Given the simplicity and sensitivity of this approach, it is anticipated that this method can be adapted to any strains of bacteria and lytic phages that are commonly used for fermentation, and has potential for a rapid detection of lytic phage contamination in the fermentation industry.

Facile generation of cell microarrays using vacuum degassing and coverslip sweeping.

A simple method to generate cell microarrays with high-percentage well occupancy and well-defined cell confinement is presented. This method uses a synergistic combination of vacuum degassing and coverslip sweeping. The vacuum degassing step dislodges air bubbles from the microwells, which in turn enables the cells to enter the microwells, while the physical sweeping step using a glass coverslip removes the excess cells outside the microwells. This low-cost preparation method provides a simple solution to generating cell microarrays that can be performed in basic research laboratories and point-of-care settings for routine cell-based screening assays.

Rapid assessment of drug response in cancer cells using microwell array and molecular imaging.

Selection of personalized chemotherapy regimen for individual patients has significant potential to improve chemotherapy efficacy and to reduce the deleterious effects of ineffective chemotherapy drugs. In this study, a rapid and high-throughput in vitro drug response assay was developed using a combination of microwell array and molecular imaging. The microwell array provided high-throughput analysis of drug response, which was quantified based on the reduction in intracellular uptake (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose) (2-NBDG). Using this synergistic approach, the drug response measurement was completed within 4 h, and only a couple thousand cells were needed for quantification. The broader application of this microwell molecular imaging approach was demonstrated by evaluating the drug response of two cancer cell lines, cervical (HeLa) and bladder (5637) cancer cells, to two distinct classes of chemotherapy drugs (cisplatin and paclitaxel). This approach did not require an extended cell culturing period, and the quantification of cellular drug response was 4-16 times faster compared with other cell-microarray drug response studies. Moreover, this molecular imaging approach had comparable sensitivity to traditional cell viability assays, i.e., the MTT assay and propidium iodide labeling of cellular nuclei;and similar throughput results as flow cytometry using only 1,000-2,000 cells. Given the simplicity and robustness of this microwell molecular imaging approach, it is anticipated that the assay can be adapted to quantify drug responses in a wide range of cancer cells and drugs and translated to clinical settings for a rapid in vitro drug response using clinically isolated samples.

Capture and detection of T7 bacteriophages on a nanostructured interface.

A highly ordered array of T7 bacteriophages was created by the electrophoretic capture of phages onto a nanostructured array with wells that accommodated the phages. Electrophoresis of bacteriophages was achieved by applying a positive potential on an indium tin oxide electrode at the bottom of the nanowells. Nanoscale arrays of phages with different surface densities were obtained by changing the electric field applied to the bottom of the nanowells. The applied voltage was shown to be the critical factor in generating a well-ordered phage array. The number of wells occupied by a phage, and hence the concentration of phages in a sample solution, could be quantified by using a DNA intercalating dye that rapidly stains the T7 phage. The fluorescence signal was enhanced by the intrinsic photonic effect made available by the geometry of the platform. It was shown that the quantification of phages on the array was 6 orders of magnitude better than could be obtained with a fluorescent plate reader. The device opens up the possibility that phages can be detected directly without enrichment or culturing, and by detecting phages that specifically infect bacteria of interest, rapid pathogen detection becomes possible.

Cellulose nanofibrils improve dispersibility and stability of silver nanoparticles and induce production of bacterial extracellular polysaccharides.

Many polymer-stabilized silver nanoparticles (AgNPs) with enhanced antibacterial properties have been synthesized, but very little is known about the fate of these materials and their interactions with microbes in physiological solutions. In this study, we evaluated the role of cellulose nanofibrils (CNFs) in stabilizing AgNPs (CNF-AgNPs) in a bacterial growth medium, determined the antibacterial properties of CNF-AgNPs and assessed the CNF and CNF-AgNP interactions with bacteria. The attachment of AgNPs to CNFs significantly improved the stability and dispersibility of AgNPs in the bacterial growth medium compared to colloidal AgNPs. The CNF-AgNPs exerted a concentration-dependent growth-inhibitory effect on E. coli. An extracellular polysaccharide (EPS)-like structure was formed around the E. coli bacterium when incubated with a sub-lethal CNF-AgNP concentration, which led to the clustering of neighboring bacteria. No EPS-like structure was observed around the bacterium incubated with high concentration of CNF-AgNPs. Overall, this study demonstrated that CNFs significantly improved the stability and dispersibility of AgNPs in physiological medium, validated the antimicrobial potential of CNF-AgNPs, and provided an insight into the physio-chemical interactions between bacteria and CNF-AgNPs in a physiological growth medium.

Direct visualization of electrophoretic mobility shift assays using nanoparticle-aptamer conjugates.

Here, we demonstrate that aptamers tethered to gold nanoparticles enable direct visualization of protein-oligonucleotide interactions during gel electrophoresis. This technique is used to confirm that an aptamer previously identified as binding to C-reactive protein (CRP) only binds to the monomeric form of CRP. While native, pentameric CRP (pCRP) is used in clinical assays to predict cardiovascular disease (CVD) risk, it is the monomeric isoform that is more strongly associated with pro-inflammatory and pro-atherogenic effects. To visualize this selectivity, the CRP-aptamer was conjugated to streptavidin-coated gold nanoparticles and the mobility of the free oligonucleotide-nanoparticle conjugate (ON-NP) and the protein/ON-NP complex bands were visualized and recorded during electrophoresis using a simple digital camera. At a concentration of 6 µg/mL, monomeric CRP showed a significant decrease in the observed ON-NP mobility, whereas no change in mobility was observed with pCRP up to 18 µg/mL. Advantages of this nanoparticle-based electrophoretic mobility shift assay (NP-EMSA) over the traditional EMSA include real-time detection of protein-oligonucleotide interactions, the avoidance of harmful radioisotopes, and elimination of the need for expensive gel imagers. The availability of both the NP-EMSA technique and an mCRP-specific probe will allow for improved clinical diagnostic to more accurately predict future CVD risk.

Membrane curvature recognition by C-reactive protein using lipoprotein mimics.

It has been reported that the oxidation of phosphatidylcholine (PC) is necessary for C-reactive protein (CRP) to bind to lipid membranes, but it remains elusive why CRP only binds oxidized membranes. Here we offer a new perspective on the role of membrane curvature and CRP binding using engineered lipoprotein particle (LPP) mimics. We show that CRP binds preferentially to LPP mimics with diameters of ≤ 28 nm, and binding of CRP to these mimics leads to the dissociation of native CRP into monomeric CRP, exposing CRP neo-epitopes that bind C1q. We also show that the smaller LPP mimics compete for CRP binding to oxidized low density lipoproteins (oxLDLs), suggesting that these mimics expose the same PC epitopes as those found on oxLDLs. Results from this study suggest that membrane curvature could be an additional factor influencing CRP binding of damaged membranes distinct from the oxidation of PC lipids.

C-reactive protein (CRP) aptamer binds to monomeric but not pentameric form of CRP.

Native C-reactive protein (CRP) is composed of five identical subunits arranged in a pentameric structure (pCRP). Binding of pCRP to damaged cell membranes produces a second isoform, modified CRP, which has similar antigenicity to isolated monomeric subunits of CRP (mCRP). Emerging evidence indicates that modified CRP plays a role in inflammation and atherosclerosis, however, there are very few techniques that can distinguish the different isoforms of CRP. Here we show that an RNA aptamer binds specifically to mCRP and not to pCRP. Using this aptamer, we describe a simple, fast, and sensitive assay to detect nanomolar concentrations of mCRP using fluorescence anisotropy. In addition, we show that this aptamer can be used to detect mCRP in polyacrylamide gels and bound to a surface using total internal reflection fluorescence microscopy. The biological activity of the mCRP we prepared by heating pCRP with 0.1% sodium dodecyl sulfate was confirmed by observing binding to the complement protein, C1q. This probe provides an important tool for CRP research and has the potential to improve clinical diagnostics that predict risk for cardiovascular disease.

Electrophoretic Mobility of Lipoprotein Nanoparticle Mimics.

Lipoprotein particles (LPPs) are biological nanoparticles whose physiological roles are greatly influenced by their sizes. The four major classes of LP are: very low density lipoprotein, intermediate density lipoprotein, low density lipoprotein (LDL) and high density lipoprotein. Since the predominance of small, dense LDLs is associated with increased risk of coronary artery disease (CAD) and diabetes mellitus, LPP profiling can be used to predict metabolic risk factors. Highly tunable LPP mimics can be synthesized using nanoparticles to carefully control for size, lipid composition and surface charge to facilitate the study LPPs in CAD. Here, we engineered LPP mimics using gold nanoparticles between 10-50 nm in diameters. We measured the mobility and zeta potential of these LPP mimics and showed that each mimics have distinct electrokinetic properties and are electrostatically stable.

Curcumin reduces alpha-synuclein induced cytotoxicity in Parkinson's disease cell model.

BACKGROUND: Overexpression and abnormal accumulation of aggregated alpha-synuclein (alphaS) have been linked to Parkinson's disease (PD) and other synucleinopathies. alphaS can misfold and adopt a variety of morphologies but recent studies implicate oligomeric forms as the most cytotoxic species. Both genetic mutations and chronic exposure to neurotoxins increase alphaS aggregation and intracellular reactive oxygen species (ROS), leading to mitochondrial dysfunction and oxidative damage in PD cell models. RESULTS: Here we show that curcumin can alleviate alphaS-induced toxicity, reduce ROS levels and protect cells against apoptosis. We also show that both intracellular overexpression of alphaS and extracellular addition of oligomeric alphaS increase ROS which induces apoptosis, suggesting that aggregated alphaS may induce similar toxic effects whether it is generated intra- or extracellulary. CONCLUSIONS: Since curcumin is a natural food pigment that can cross the blood brain barrier and has widespread medicinal uses, it has potential therapeutic value for treating PD and other neurodegenerative disorders.

Antifibrillizing agents catalyze the formation of unstable intermediate aggregates of beta-amyloid.

Although Alzheimer's disease (AD) is characterized by the extracellular deposition of fibrillar aggregates of beta-amyloid (Abeta), transient oligomeric species of Abeta are increasingly implicated in the pathogenesis of AD. Natively unfolded monomeric Abeta can misfold and progressively assemble into fibrillar aggregates, following a well-established "on pathway" seeded-nucleation mechanism. Here, we show that three simple saccharides, mannose, sucrose, and raffinose, alter Abeta aggregation kinetics and morphology. The saccharides inhibit formation of Abeta fibrils but promote formation of various oligomeric aggregate species through different "off pathway" aggregation mechanisms at 37 degrees C but not at 60 degrees C. The various oligomeric Abeta aggregates formed when coincubated with the different saccharides are morphologically distinct but all are toxic toward SH-SY5Y human neuroblastoma cells, increasing the level of toxicity and greatly prolonging toxicity compared with Abeta alone. As a wide variety of anti-Abeta aggregation strategies are being actively pursued as potential therapeutics for AD, these studies suggest that care must be taken to ensure that the therapeutic agents also block toxic oligomeric Abeta assembly as well as inhibit fibril formation.

Conformational targeting of fibrillar polyglutamine proteins in live cells escalates aggregation and cytotoxicity.

BACKGROUND: Misfolding- and aggregation-prone proteins underlying Parkinson's, Huntington's and Machado-Joseph diseases, namely alpha-synuclein, huntingtin, and ataxin-3 respectively, adopt numerous intracellular conformations during pathogenesis, including globular intermediates and insoluble amyloid-like fibrils. Such conformational diversity has complicated research into amyloid-associated intracellular dysfunction and neurodegeneration. To this end, recombinant single-chain Fv antibodies (scFvs) are compelling molecular tools that can be selected against specific protein conformations, and expressed inside cells as intrabodies, for investigative and therapeutic purposes. METHODOLOGY/PRINCIPAL FINDINGS: Using atomic force microscopy (AFM) and live-cell fluorescence microscopy, we report that a human scFv selected against the fibrillar form of alpha-synuclein targets isomorphic conformations of misfolded polyglutamine proteins. When expressed in the cytoplasm of striatal cells, this conformation-specific intrabody co-localizes with intracellular aggregates of misfolded ataxin-3 and a pathological fragment of huntingtin, and enhances the aggregation propensity of both disease-linked polyglutamine proteins. Using this intrabody as a tool for modulating the kinetics of amyloidogenesis, we show that escalating aggregate formation of a pathologic huntingtin fragment is not cytoprotective in striatal cells, but rather heightens oxidative stress and cell death as detected by flow cytometry. Instead, cellular protection is achieved by suppressing aggregation using a previously described intrabody that binds to the amyloidogenic N-terminus of huntingtin. Analogous cytotoxic results are observed following conformational targeting of normal or polyglutamine-expanded human ataxin-3, which partially aggregate through non-polyglutamine domains. CONCLUSIONS/SIGNIFICANCE: These findings validate that the rate of aggregation modulates polyglutamine-mediated intracellular dysfunction, and caution that molecules designed to specifically hasten aggregation may be detrimental as therapies for polyglutamine disorders. Moreover, our findings introduce a novel antibody-based tool that, as a consequence of its general specificity for fibrillar conformations and its ability to function intracellularly, offers broad research potential for a variety of human amyloid diseases.

Cyclodextrins promote protein aggregation posing risks for therapeutic applications.

The presence of neurofibrillary tangles (NFTs) is a hallmark feature of various neurodegenerative disorders including Alzheimer's (AD) and Niemann-Pick type C (NPC) diseases. NFTs have been correlated with elevated cholesterol levels and a cholesterol-scavenging compound, cyclodextrin, effectively modulates and traffics cholesterol from cell bodies in NPC disease models. Cyclodextrins are also used as drug carriers to the blood-brain barrier (BBB) and other tissues. While cyclodextrins have potential value in treating brain diseases, it is important to determine how cyclodextrins affect natively unfolded proteins such as beta-amyloid (Abeta) whose aggregation has been correlated with AD. We show that cyclodextrins drastically alter Abeta aggregation kinetics and induce morphological changes to Abeta that can enhance toxicity towards SH-SY5Y human neuroblastoma cells. These results suggest that care must be taken when using cyclodextrins for BBB delivery or for treatment of brain disease because cyclodextrins can promote toxic aggregation of Abeta.

Detecting morphologically distinct oligomeric forms of alpha-synuclein.

Neuropathologic and genetics studies as well as transgenic animal models have provided strong evidence linking misfolding and aggregation of alpha-synuclein to the progression of Parkinson disease (PD) and other related disorders. A growing body of evidence implicates various oligomeric forms of alpha-synuclein as the toxic species responsible for neurodegeneration and neuronal cell death. Although numerous different oligomeric forms of alpha-synuclein have been identified in vitro, it is not known which forms are involved in PD or how, when, and where different forms contribute to the progression of PD. Reagents that can interact with specific aggregate forms of alpha-synuclein would be very useful not only as tools to study how different aggregate forms affect cell function, but also as potential diagnostic and therapeutic agents for PD. Here we show that a single chain antibody fragment (syn-10H scFv) isolated from a phage display antibody library binds to a larger, later stage oligomeric form of alpha-synuclein than a previously reported oligomeric specific scFv isolated in our laboratory. The scFv described here inhibits aggregation of alpha-synuclein in vitro, blocks extracellular alpha-synuclein-induced toxicity in both undifferentiated and differentiated human neuroblastoma cell lines (SH-SY5Y), and specifically recognizes naturally occurring aggregates in PD but not in healthy human brain tissue.

Characterizing antibody specificity to different protein morphologies by AFM.

Protein misfolding and aggregation can lead to several neurodegenerative diseases including Alzheimer's Disease (AD), Parkinson's Disease (PD) and Huntington's Disease (HD). While the respective proteins involved in each disease differ in their pathological effects and amino acid sequences, the aggregated forms all share a common cross beta-sheet conformation. Substantial controversy exists over the roles of the different aggregate morphologies in disease onset and progression, and analytical tools such as morphology specific antibodies are needed to distinguish between the different protein morphologies in situ. Here we utilize atomic force microscopy (AFM) to characterize the binding of three single chain antibody fragments (scFvs) to different morphologies of alpha-synuclein (alphaS). From the topographic images generated using the AFM, we were able to show that one scFv bound all morphologies of alphaS, a second bound only oligomeric alphaS, and a third bound only fibrillar alphaS by comparing the height distribution of the different alphaS morphologies with and without addition of the different scFvs. These results demonstrate the versatility of the AFM-based technique as an easy tool to characterize specific antigen-antibody binding and the potential applications of scFvs as promising immunodiagnostics for protein misfolding diseases.

Isolation of a human single chain antibody fragment against oligomeric alpha-synuclein that inhibits aggregation and prevents alpha-synuclein-induced toxicity.

Protein misfolding and aggregation are pathological aspects of numerous neurodegenerative diseases. Aggregates of alpha-synuclein are major components of the Lewy bodies and Lewy neurites associated with Parkinson's Disease (PD). A natively unfolded protein, alpha-synuclein can adopt different aggregated morphologies, including oligomers, protofibrils and fibrils. The small oligomeric aggregates have been shown to be particularly toxic. Antibodies that neutralize the neurotoxic aggregates without interfering with beneficial functions of monomeric alpha-synuclein can be useful therapeutics. We were able to isolate single chain antibody fragments (scFvs) from a phage displayed antibody library against the target antigen morphology using a novel biopanning technique that utilizes atomic force microscopy (AFM) to image and immobilize specific morphologies of alpha-synuclein. The scFv described here binds only to an oligomeric form of alpha-synuclein and inhibits both aggregation and toxicity of alpha-synuclein in vitro. This scFv can have potential therapeutic value in controlling misfolding and aggregation of alpha-synuclein in vivo when expressed intracellularly in dopaminergic neurons as an intrabody.

Spatiotemporal localization of injury potentials in DRG neurons during vincristine-induced axonal degeneration.

The distal to proximal degeneration of axons, or "dying back" is a common pattern of neuropathology in many diseases of the PNS and CNS. A long-standing debate has centered on whether this pattern of neurodegeneration is due to an insult to the cell body or to the axon itself, although it is likely that mechanisms are different for specific disease entities. We have addressed this question in a model system of vincristine-induced axonal degeneration. Here, we created a novel experimental apparatus combining a microfluidic divider with a multielectrode array substrate to allow for independent monitoring of injury-induced electrical activity from dorsal root ganglion (DRG) cell bodies and axons while isolating them into their own culture microenvironments. At specified doses, exposure of the cell body to vincristine caused neither morphological neurodegeneration nor persistent hyperexcitability. In comparison, exposure of the distal axon to the same dose of vincristine first caused a decrease in the excitability of the axon and then axonal degeneration in a dying back pattern. Additionally, exposure of axons to vincristine caused an initial period of hyperexcitability in the cell bodies, suggesting that a signal is transmitted from the distal axon to the soma during the progression of vincristine-induced axonal degeneration. These data support the proposition that vincristine has a direct neurotoxic effect on the axon.

A compartmented neuronal culture system in microdevice format.

This paper describes a microfabricated compartmented culture system (mu-CCS) for studying the effects of drugs on cultured neurons. We describe the fabrication of the microsystem and show the ability to culture DRG neurons in the microsystem. Furthermore, we demonstrate the ability to culture neurons with extensions growing into adjoining compartments while maintaining fluid isolation between compartments. The axonal growth pattern was controlled along the surface of the glass microelectrode substrate using a micropatterned collagen film. The ability to isolate fluids to selected compartments while simultaneously allowing intercompartmental growth of the axons enables various studies in which selected segments of neurons or populations of neurons can be selectively exposed to biochemical treatment. The neurotoxin vincristine was used as the test vehicle to demonstrate the functionality of the mu-CCS. Vincristine was applied to the axonal compartment to show that the interaction of drugs with DRG neurons progresses in a way similar to neurons cultured/exposed using conventional techniques.