Oxidation of Hydrogen Sulfide to Polysulfide and Thiosulfate by a Carbon Nanozyme: Therapeutic Implications with an Emphasis on Down Syndrome.

Hydrogen sulfide (H2 S) is a noxious, potentially poisonous, but necessary gas produced from sulfur metabolism in humans. In Down Syndrome (DS), the production of H2 S is elevated and associated with degraded mitochondrial function. Therefore, removing H2 S from the body as a stable oxide could be an approach to reducing the deleterious effects of H2 S in DS. In this report we describe the catalytic oxidation of hydrogen sulfide (H2 S) to polysulfides (HS2+n - ) and thiosulfate (S2 O3 2- ) by poly(ethylene glycol) hydrophilic carbon clusters (PEG-HCCs) and poly(ethylene glycol) oxidized activated charcoal (PEG-OACs), examples of oxidized carbon nanozymes (OCNs). We show that OCNs oxidize H2 S to polysulfides and S2 O3 2- in a dose-dependent manner. The reaction is dependent on O2 and the presence of quinone groups on the OCNs. In DS donor lymphocytes we found that OCNs increased polysulfide production, proliferation, and afforded protection against additional toxic levels of H2 S compared to untreated DS lymphocytes. Finally, in Dp16 and Ts65DN murine models of DS, we found that OCNs restored osteoclast differentiation. This new action suggests potential facile translation into the clinic for conditions involving excess H2 S exemplified by DS.

Oxidized Activated Charcoal Nanozymes: Synthesis, and Optimization for In Vitro and In Vivo Bioactivity for Traumatic Brain Injury.

Carbon-based superoxide dismutase (SOD) mimetic nanozymes have recently been employed as promising antioxidant nanotherapeutics due to their distinct properties. The structural features responsible for the efficacy of these nanomaterials as antioxidants are, however, poorly understood. Here, the process-structure-property-performance properties of coconut-derived oxidized activated charcoal (cOAC) nano-SOD mimetics are studied by analyzing how modifications to the nanomaterial's synthesis impact the size, as well as the elemental and electrochemical properties of the particles. These properties are then correlated to the in vitro antioxidant bioactivity of poly(ethylene glycol)-functionalized cOACs (PEG-cOAC). Chemical oxidative treatment methods that afford smaller, more homogeneous cOAC nanoparticles with higher levels of quinone functionalization show enhanced protection against oxidative damage in bEnd.3 murine endothelioma cells. In an in vivo rat model of mild traumatic brain injury (mTBI) and oxidative vascular injury, PEG-cOACs restore cerebral perfusion rapidly to the same extent as the former nanotube-derived PEG-hydrophilic carbon clusters (PEG-HCCs) with a single intravenous injection. These findings provide a deeper understanding of how carbon nanozyme syntheses can be tailored for improved antioxidant bioactivity, and set the stage for translation of medical applications.

The Effects of Antioxidant Nutraceuticals on Cellular Sulfur Metabolism and Signaling.

Significance: Nutraceuticals are ingested for health benefits, in addition to their general nutritional value. These dietary supplements have become increasingly popular since the late 20th century and they are a rapidly expanding global industry approaching a half-trillion U.S. dollars annually. Many nutraceuticals are promulgated as potent antioxidants. Recent Advances: Experimental support for the efficacy of nutraceuticals has lagged behind anecdotal exuberance. However, accumulating epidemiological evidence and recent, well-controlled clinical trials are beginning to support earlier animal and in vitro studies. Although still somewhat limited, encouraging results have been suggested in essentially all organ systems and against a wide range of pathophysiological conditions. Critical Issues: Health benefits of "antioxidant" nutraceuticals are largely attributed to their ability to scavenge oxidants. This has been criticized based on several factors, including limited bioavailability, short tissue retention time, and the preponderance of endogenous antioxidants. Recent attention has turned to nutraceutical activation of downstream antioxidant systems, especially the Keap1/Nrf2 (Kelch like ECH associated protein 1/nuclear factor erythroid 2-related factor 2) axis. The question now becomes, how do nutraceuticals activate this axis? Future Directions: Reactive sulfur species (RSS), including hydrogen sulfide (H2S) and its metabolites, are potent activators of the Keap1/Nrf2 axis and avid scavengers of reactive oxygen species. Evidence is beginning to accumulate that a variety of nutraceuticals increase cellular RSS by directly providing RSS in the diet, or through a number of catalytic mechanisms that increase endogenous RSS production. We propose that nutraceutical-specific targeting of RSS metabolism will lead to the design and development of even more efficacious antioxidant therapeutic strategies. Antioxid. Redox Signal. 38, 68-94.

Naphthoquinones Oxidize H2S to Polysulfides and Thiosulfate, Implications for Therapeutic Applications.

1,4-Napththoquinones (NQs) are clinically relevant therapeutics that affect cell function through production of reactive oxygen species (ROS) and formation of adducts with regulatory protein thiols. Reactive sulfur species (RSS) are chemically and biologically similar to ROS and here we examine RSS production by NQ oxidation of hydrogen sulfide (H2S) using RSS-specific fluorophores, liquid chromatography-mass spectrometry, UV-Vis absorption spectrometry, oxygen-sensitive optodes, thiosulfate-specific nanoparticles, HPLC-monobromobimane derivatization, and ion chromatographic assays. We show that NQs, catalytically oxidize H2S to per- and polysulfides (H2Sn, n = 2−6), thiosulfate, sulfite and sulfate in reactions that consume oxygen and are accelerated by superoxide dismutase (SOD) and inhibited by catalase. The approximate efficacy of NQs (in decreasing order) is, 1,4-NQ ≈ juglone ≈ plumbagin > 2-methoxy-1,4-NQ ≈ menadione >> phylloquinone ≈ anthraquinone ≈ menaquinone ≈ lawsone. We propose that the most probable reactions are an initial two-electron oxidation of H2S to S0 and reduction of NQ to NQH2. S0 may react with H2S or elongate H2Sn in variety of reactions. Reoxidation of NQH2 likely involves a semiquinone radical (NQ·−) intermediate via several mechanisms involving oxygen and comproportionation to produce NQ and superoxide. Dismutation of the latter forms hydrogen peroxide which then further oxidizes RSS to sulfoxides. These findings provide the chemical background for novel sulfur-based approaches to naphthoquinone-directed therapies.

Pancreatic tumor microenvironmental acidosis and hypoxia transform gold nanorods into cell-penetrant particles for potent radiosensitization.

Coating nanoparticles with stealth epilayers increases circulation time by evading opsonization, macrophage phagocytosis, and reticuloendothelial sequestration. However, this also reduces internalization by cancer cells upon reaching the tumor. We designed gold nanorods (GNRs) with an epilayer that retains stealth properties in circulation but transforms spontaneously in the acidotic tumor microenvironment to a cell-penetrating particle. We used a customized stoichiometric ratio of l-glutamic acid and l-lysine within an amphiphilic polymer of poly(l-glutamic acid-co-l-lysine), or P(Glu-co-Lys), to effect this transformation in acidotic environments. P(Glu-co-Lys)-GNRs were internalized by cancer cells to facilitate potent in vitro radiosensitization. When administered intravenously in mice, they accumulate in the periphery and core of tumors without any signs of serum biochemical or hematological alterations, normal organ histopathological abnormalities, or overt deterioration in animal health. Furthermore, P(Glu-co-Lys)-GNRs penetrated the tumor microenvironment to accumulate in the hypoxic cores of tumors to potently radiosensitize heterotopic and orthotopic pancreatic cancers in vivo.

Uncloaking cell-impermeant gold nanorods via tumor microenvironmental cathepsin B facilitates cancer cell penetration and potent radiosensitization.

Major impediments to conveyance of intravenously administered drugs to tumors are biofouling, opsonization, and rapid clearance from the circulation by macrophages and reticuloendothelial phagocytes. Cloaking nanoparticles with stealth epilayers partly overcomes these hurdles but it also foils interactions with tumor cells. Here, we describe the synthesis, characterization, and validation of smart gold nanorods (GNRs) that spontaneously transform from inert passengers in the blood stream to active cell-penetrating nanoparticles within tumors to potently sensitize tumors to radiation therapy. Intrinsically cationic and cell-penetrating GNRs were shielded from phagocytosis with a cloaking polyethylene glycol epilayer containing an intervening cleavable peptide. In the absence of an external trigger, this epilayer is clipped off by the tumor microenvironmental protease, cathepsin B, in colorectal cancers to uncloak and expose the free-circulating native unPEGylated GNR that is readily internalized by cancer cells and turn into immovable small clusters of GNRs. Selective uncloaking of GNRs in the tumor reduced off-target toxicity confirmed by hematologic, biochemical, and histopathological analysis of blood, serum, and normal organs, respectively. Subsequent irradiation led to significant tumor growth delay and improved survival of mice. By addressing multiple barriers to efficient transport and cellular internalization of nanoparticles, our results demonstrate that clinically meaningful radiosensitization can be achieved with rationally designed GNRs.

Coenzyme Q10 and related quinones oxidize H2S to polysulfides and thiosulfate.

In the canonical pathway for mitochondrial H2S oxidation electrons are transferred from sulfide:quinone oxidoreductase (SQR) to complex III via ubiquinone (CoQ10). We previously observed that a number of quinones directly oxidize H2S and we hypothesize that CoQ10 may have similar properties. Here we examine H2S oxidation by CoQ10 and more hydrophilic, truncated forms, CoQ1 and CoQ0, in buffer using H2S and polysulfide fluorophores (AzMC and SSP4), silver nanoparticles to measure thiosulfate (H2S2O3), mass spectrometry to identify polysulfides and O2-sensitive optodes to measure O2 consumption. We show that all three quinones concentration-dependently catalyze the oxidization of H2S to polysulfides and thiosulfate in buffer with the potency CoQ0>CoQ1>CoQ10 and that CoQ0 specifically oxidizes H2S to per-polysulfides, H2S2,3,4. These reactions consume and require oxygen and are augmented by addition of SOD suggesting that the quinones, not superoxide, oxidize H2S. Related quinones, MitoQ, menadione and idebenone, oxidize H2S in similar reactions. Exogenous CoQ0 decreases cellular H2S and increases polysulfides and thiosulfate production and this is also O2-dependent, suggesting that the quinone has similar effects on sulfur metabolism in cells. Collectively, these results suggest an additional endogenous mechanism for H2S metabolism and a potential therapeutic approach in H2S-related metabolic disorders.

The Chemical Basis of Intracerebral Hemorrhage and Cell Toxicity With Contributions From Eryptosis and Ferroptosis.

Intracerebral hemorrhage (ICH) is a particularly devastating event both because of the direct injury from space-occupying blood to the sequelae of the brain exposed to free blood components from which it is normally protected. Not surprisingly, the usual metabolic and energy pathways are overwhelmed in this situation. In this review article, we detail the complexity of red blood cell degradation, the contribution of eryptosis leading to hemoglobin breakdown into its constituents, the participants in that process, and the points at which injury can be propagated such as elaboration of toxic radicals through the metabolism of the breakdown products. Two prominent products of this breakdown sequence, hemin, and iron, induce a variety of pathologies including free radical damage and DNA breakage, which appear to include events independent from typical oxidative DNA injury. As a result of this confluence of damaging elements, multiple pathways of injury, cell death, and survival are likely engaged including ferroptosis (which may be the same as oxytosis but viewed from a different perspective) and senescence, suggesting that targeting any single cause will likely not be a sufficient strategy to maximally improve outcome. Combination therapies in addition to safe methods to reduce blood burden should be pursued.

Pervasive Genomic Damage in Experimental Intracerebral Hemorrhage: Therapeutic Potential of a Mechanistic-Based Carbon Nanoparticle.

Therapy for intracerebral hemorrhage (ICH) remains elusive, in part dependent on the severity of the hemorrhage itself as well as multiple deleterious effects of blood and its breakdown products such as hemin and free iron. While oxidative injury and genomic damage have been seen following ICH, the details of this injury and implications remain unclear. Here, we discovered that, while free iron produced mostly reactive oxygen species (ROS)-related single-strand DNA breaks, hemin unexpectedly induced rapid and persistent nuclear and mitochondrial double-strand breaks (DSBs) in neuronal and endothelial cell genomes and in mouse brains following experimental ICH comparable to that seen with γ radiation and DNA-complexing chemotherapies. Potentially as a result of persistent DSBs and the DNA damage response, hemin also resulted in senescence phenotype in cultured neurons and endothelial cells. Subsequent resistance to ferroptosis reported in other senescent cell types was also observed here in neurons. While antioxidant therapy prevented senescence, cells became sensitized to ferroptosis. To address both senescence and resistance to ferroptosis, we synthesized a modified, catalytic, and rapidly internalized carbon nanomaterial, poly(ethylene glycol)-conjugated hydrophilic carbon clusters (PEG-HCC) by covalently bonding the iron chelator, deferoxamine (DEF). This multifunctional nanoparticle, DEF-HCC-PEG, protected cells from both senescence and ferroptosis and restored nuclear and mitochondrial genome integrity in vitro and in vivo. We thus describe a potential molecular mechanism of hemin/iron-induced toxicity in ICH that involves a rapid induction of DSBs, senescence, and the consequent resistance to ferroptosis and provide a mechanistic-based combinatorial therapeutic strategy.

Revisiting the intersection of amyloid, pathologically modified tau and iron in Alzheimer's disease from a ferroptosis perspective.

The complexity of Alzheimer's disease (AD) complicates the search for effective treatments. While the key roles of pathologically modified proteins has occupied a central role in hypotheses of the pathophysiology, less attention has been paid to the potential role for transition metals overload, subsequent oxidative stress, and tissue injury. The association of transition metals, the major focus heretofore iron and amyloid, the same can now be said for the likely pathogenic microtubular associated tau (MAPT). This review discusses the interplay between iron, pathologically modified tau and oxidative stress, and connects many related discoveries. Basic principles of the transition to pathological MAPT are discussed. Iron, its homeostatic mechanisms, the recently described phenomenon of ferroptosis and purported, although still controversial roles in AD are reviewed as well as considerations to overcome existing hurdles of iron-targeted therapeutic avenues that have been attempted in AD. We summarize the involvement of multiple pathological pathways at different disease stages of disease progression that supports the potential for a combinatorial treatment strategy targeting multiple factors.

Critical Comparison of the Superoxide Dismutase-like Activity of Carbon Antioxidant Nanozymes by Direct Superoxide Consumption Kinetic Measurements.

The superoxide dismutase-like activity of poly(ethylene glycolated) hydrophilic carbon clusters (PEG-HCCs), anthracite and bituminous graphene quantum dots (PEG-aGQDs and PEG-bGQDs, respectively), and two fullerene carbon nanozymes, tris malonyl-C60 fullerene (C3) and polyhydroxylated-C60 fullerene (C60-OHn), were compared using direct optical stopped-flow kinetic measurements, together with three native superoxide dismutases (SODs), CuZnSOD, MnSOD, and FeSOD, at both pH 12.7 and 8.5. Computer modeling including both SOD catalytic steps and superoxide self-dismutation enabled the best choice of catalyst concentration with minimal contribution to the observed kinetic change from the substrate self-dismutation. Biexponential fitting to the kinetic data ranks the rate constant (M-1 s-1) in the order of PEG-HCCs > CuZnSOD ≈ MnSOD ≈ PEG-aGQDs ≈ PEG-bGQDs > FeSOD ≫ C3 > C60-OHn at pH 12.7 and MnSOD > CuZnSOD ≈ PEG-HCCs > FeSOD > PEG-aGQDs ≈ PEG-bGQDs ≫ C3 ≈ C60-OHn at pH 8.5. Nonlinear regression of the kinetic model above yielded the same ranking as the biexponential fit, but provided better mechanistic insight. The data obtained by freeze-quench EPR direct assay at pH 12.7 also yield the same ranking as stopped-flow data. This is a necessary assessment of a panel of proclaimed carbon nano SOD mimetics using the same two direct methods, revealing a dramatic, 3-4 orders of magnitude difference in SOD activity between PEG-HCCs/PEG-GQDs from soluble fullerenes.

Improving the Shape Yield and Long-Term Stability of Gold Nanoprisms with Poly(vinylpyrrolidone).

Gold nanoprisms (AuNPRs) are anisotropic nanostructures that have gained great attention in recent years because of their interesting and unique optical properties that can be tailored for biomedical, energy, and sensing applications. At present, several protocols have reported the high yield synthesis of AuNPRs of different dimensions using a seed-mediated approach. However, there is a need to develop reproducible and scalable methods with the goal of a controllable synthesis. Here, we report an improved seed-mediated synthesis of small monodisperse AuNPRs of distinct sizes in high yield using poly(vinylpyrrolidone) (PVP) as an additive in nanomolar concentrations. We show optimal synthetic parameters for a blue-shifting of the surface plasmon resonance band which correlates with the reduction in the edge length (L) of AuNPRs from 75 to 35 nm. Using measured extinction coefficients for AuNPRs of different sizes, a linear equation is proposed to estimate the concentration of unknown samples by using Beer's law. Interestingly, the use of nanomolar amounts of PVP during the growth of AuNPRs significantly improves the shape yield. The surface chemistry properties of AuNPRs were measured by X-ray photoelectron spectroscopy and attenuated total reflectance infrared spectroscopy and revealed that PVP chains interact with AuNPRs through the carbonyl oxygen. This method is reproducible and scalable and enables the synthesis of AuNPRs with long-term shape stability (1 year) in aqueous solution.

Catalytic oxidation and reduction reactions of hydrophilic carbon clusters with NADH and cytochrome C: features of an electron transport nanozyme.

Previously, our group reported on the promising efficacy of poly(ethylene glycol)-hydrophilic carbon clusters (PEG-HCCs) to work as broadly active and high capacity antioxidants in brain ischemia and injury models including stroke and traumatic brain injury coupled with hemorrhagic shock. PEG-HCCs are a carbon nanomaterial derived from harsh oxidation of single wall carbon nanotubes and covalently modified with poly(ethylene glycol). They retain no tubular remnants and are composed of a highly oxidized carbon core functionalized with epoxy, peroxyl, quinone, ketone, carboxylate, and hydroxyl groups. HCCs are the redox active carbon core of PEG-HCCs, which have a broad reduction potential range starting at +200 mV and extending to -2 V. Here we describe a new property of these materials: the ability to catalytically transfer electrons between key surrogates and proteins of the mitochondrial electron transport complex in a catalytic fashion consistent with the concept of a nanozyme. The estimated reduction potential of PEG-HCCs is similar to that of ubiquinone and they enabled the catalytic transfer of electrons from low reduction potential species to higher reduction electron transport complex constituents. PEG-HCCs accelerated the reduction of resazurin (a test indicator of mitochondrial viability) and cytochrome c by NADH and ascorbic acid in solution. Kinetic experiments suggested a transient tertiary complex. Electron paramagnetic resonance demonstrated NADH increased the magnitude of PEG-HCCs' intrinsic radical, which then reduced upon subsequent addition of cytochrome c or resazurin. Deconvolution microscopy identified PEG-HCCs in close proximity to mitochondria after brief incubation with cultured SHSY-5Y human neuroblastoma cells. Compared to methylene blue (MB), considered a prototypical small molecule electron transport shuttle, PEG-HCCs were more protective against toxic effects of hydrogen peroxide in vitro and did not demonstrate impaired cell viability as did MB. PEG-HCCs were protective in vitro when cells were exposed to sodium cyanide, a mitochondrial complex IV poison. Because mitochondria are a major source of free radicals in pathology, we suggest that this newly described nanozyme action helps explain their in vivo efficacy in a range of injury models. These findings may also extend their use to mitochondrial disorders.

Highly Oxidized Graphene Quantum Dots from Coal as Efficient Antioxidants.

Graphene quantum dots (GQDs) have recently been employed in various fields including medicine as antioxidants, primarily because of favorable biocompatibility in comparison to common inorganic quantum dots, although the structural features that lead to the biological activities of GQDs are poorly understood. Here, we report that coal-derived GQDs and their poly(ethylene glycol)-functionalized derivatives serve as efficient antioxidants, and we evaluate their electrochemical, chemical, and in vitro biological activities.

Telemedicine in Prehospital Acute Stroke Care: An Expanding Infrastructure for Treatment and Research.

See Article by Geisler et al.

Functional and Structural Improvement with a Catalytic Carbon Nano-Antioxidant in Experimental Traumatic Brain Injury Complicated by Hypotension and Resuscitation.

Hypotension worsens outcome after all severities of traumatic brain injury (TBI), with loss of cerebral autoregulation being a potential contributor. Previously, we demonstrated that intravenous injection of a high capacity catalytic antioxidant, poly(ethylene)glycol conjugated hydrophilic carbon clusters (PEG-HCCs) rapidly restored cerebral perfusion and acutely restored brain oxidative balance in a TBI model complicated by hemorrhagic hypotension without evidence of toxicity. Here, we tested whether these acute effects translated into behavioral and structural benefit. TBI was generated by a cortical contusion impactor in 38 Long Evans rats, followed by blood withdrawal to a target mean arterial pressure of 40 mm Hg. PEG-HCC (2 mg/kg) or diluent was injected intravenously 80 min later at the onset of blood resuscitation followed by another injection 2 h later (doses determined in prior studies). Performance on beam walking (performed on days 1-5) and Morris water maze (MWM) (performed on days 11-15) was tested, and lesion size was determined at the termination. PEG-HCC treatment nearly completely prevented motor dysfunction (p < 0.001 vs. diluent), improved MWM performance (p < 0.001; treatment vs. time interaction) and reduced lesion size by 61% (p = 0.054). Here we show that treatment with PEG-HCCs at a clinically realistic time point (onset of resuscitation) prevented a major portion of the neurological dysfunction induced in this TBI model, and that PEG-HCCs are candidates for additional study as a potential therapeutic agent.

Efficacy of Novel Carbon Nanoparticle Antioxidant Therapy in a Severe Model of Reversible Middle Cerebral Artery Stroke in Acutely Hyperglycemic Rats.

INTRODUCTION: While oxidative stress can be measured during transient cerebral ischemia, antioxidant therapies for ischemic stroke have been clinically unsuccessful. Many antioxidants are limited in their range and/or capacity for quenching radicals and can generate toxic intermediates overwhelming depleted endogenous protection. We developed a new antioxidant class, 40 nm × 2 nm carbon nanoparticles, hydrophilic carbon clusters, conjugated to poly(ethylene glycol) termed PEG-HCCs. These particles are high-capacity superoxide dismutase mimics, are effective against hydroxyl radical, and restore the balance between nitric oxide and superoxide in the vasculature. Here, we report the effects of PEG-HCCs administered during reperfusion after transient middle cerebral artery occlusion (tMCAO) by suture in the rat under hyperglycemic conditions. Hyperglycemia occurs in one-third of stroke patients and worsens clinical outcome. In animal models, this worsening occurs largely by accelerating elaboration of reactive oxygen species (ROS) during reperfusion. METHODS: PEG-HCCs were studied for their protective ability against hydrogen peroxide in b.End3 brain endothelial cell line and E17 primary cortical neuron cultures. In vivo, hyperglycemia was induced by streptozotocin injection 2 days before tMCAO. 58 Male Sprague-Dawley rats were analyzed. They were injected IV with PBS or PEG-HCCs (4 mg/kg 2×) at the time of recanalization after either 90- or 120-min occlusion. Rats were survived for up to 3 days, and infarct volume characteristics and neurological functional outcome (modified Bederson Score) were assessed. RESULTS: PEG-HCCs were protective against hydrogen peroxide in both culture models. In vivo improvement was found after PEG-HCCs with 90-min ischemia with reduction in infarct size (42%), hemisphere swelling (46%), hemorrhage score (53%), and improvement in Bederson score (70%) (p = 0.068-0.001). Early high mortality in the 2-h in the PBS control group precluded detailed analysis, but a trend was found in improvement in all factors, e.g., reduction in infarct volume (48%; p = 0.034) and a 56% improvement in Bederson score (p = 0.055) with PEG-HCCs. CONCLUSION: This nano-antioxidant showed some improvement in several outcome measures in a severe model of tMCAO when administered at a clinically relevant time point. Long-term studies and additional models are required to assess potential for clinical use, especially for patients hyperglycemic at the time of their stroke, as these patients have the worst outcomes.

Fluorescent labeling of endogenous platelets for intravital microscopy: Effects on platelet function.

OBJECTIVE: Monitoring endogenous platelets during intravital microscopy often involves two approaches: fluorescently labeled antibodies or genetic models of platelet-specific fluorescent protein expression. Due to limited data available on platelet functional changes induced by these methods, we compared functional effects of these methods on platelets. METHODS: Platelet aggregation to collagen and thrombin, and collagen matrix-mediated platelet adhesion/aggregation under flow were tested. We assessed platelets from mice expressing EYFP on platelets (Cre(+)), littermate controls (Cre(-)), C57BL/6 mice, and platelets from vehicle control and x-488 treatment. We utilized intravital microscopy to monitor platelets in vivo using Cre(+) mice and x-488 treatment. RESULTS: Both genetic and antibody-based approaches yielded substantial platelet-specific fluorescence. Platelets from Cre(+) and Cre(-) mice behaved similarly in aggregation and adhesion/aggregation under flow. However, they exhibited significantly enhanced aggregation and higher adhesion/aggregation as compared to platelets from C57BL/6 mice. Compared to vehicle control, x-488 platelet labeling did not induce significant functional changes in vitro. Both methods of platelet labeling provided satisfactory platelet detectability in vivo. CONCLUSIONS: x-488 antibody labeling of platelets induced less alteration of platelet function than genetic approaches under our experimental conditions and seems more suitable for monitoring of endogenous platelets.

Accelerating Gold Nanorod Synthesis with Nanomolar Concentrations of Poly(vinylpyrrolidone).

A novel modification for the seedless synthesis of gold nanorods (AuNRs) has been developed. Nanomolar concentrations of 10 kDa poly(vinylpyrrolidone) (PVP) can be introduced to a growth solution containing 25, 50, or 100 mM cetyltrimethylammonium bromide (CTAB) to significantly reduce the dimensions of AuNRs. We found that PVP accelerates the growth rate of AuNRs by more than two times that of nanorods grown in 50 and 100 mM CTAB solutions. Additionally, there is a time-dependent effect of adding PVP to the nanorod growth solution that can be utilized to tune their aspect ratio. Because the concentration of PVP is far below the concentration of HAuCl4 in the reaction mixture, PVP primarily functions not as a reducing agent, but as a capping or templating ligand to stabilize the growing nanorods. Our reproducible protocol enables the synthesis of AuNRs in high yield with tunable sizes: 45 × 6.7, 28 × 5.5, and 12 × 4.5 nm for 100, 50, and 25 mM CTAB, respectively. We estimated the number of PVP chains per nanorod in growth solutions to be around 30, which suggests that the effect on the aspect ratio is caused by a direct interaction between the AuNR surface and the PVP.