Highly Ordered Polymer Nanostructures via Solvent On-Film Annealing for Surface-Enhanced Raman Scattering.

Surface-enhanced Raman scattering (SERS) has been a useful sensing technique, in which inelastic light scattering can be significantly enhanced by absorbing molecules onto rough metal surfaces or nanoparticles. Although many methods have been developed to prepare SERS substrates, it is still highly desirable and challenging to design SERS substrates, especially with highly ordered and controlled three-dimensional (3D) structures. In this work, we develop novel SERS substrates with regular volcano-shaped polymer structures using the versatile solvent on-film annealing method. Polystyrene (PS) nanospheres are first synthesized by surfactant-free emulsion polymerization and assembled on poly(methyl methacrylate) (PMMA) films. After annealing in acetic acid vapors, PMMA chains are selectively swollen and wet the surfaces of the PS nanospheres. By selectively removing the PS nanospheres using cyclohexane, volcano-shaped PMMA films can be obtained. Compared with flat PMMA films with water contact angles of ∼74°, volcano-shaped PMMA films exhibit higher water contact angles of ∼110° due to the sharp features and rough surfaces. The volcano-shaped PMMA films are then coated with gold nanoparticles (AuNPs) as SERS substrates. Using rhodamine 6G as the probe molecules, the SERS results show that the Raman signals of the volcano-shaped PMMA/AuNP hybrid substrates are much higher than those of the pristine PMMA films and PMMA films with AuNPs. For the volcano-shaped PMMA/AuNP hybrid substrates using 400 nm PS nanospheres, a high enhancement factor (EF) value of ∼1.12 × 105 with a detection limit of 10-8 M is obtained in a short integration time of 1 s. A linear calibration line with an R2 value of 0.918 is also established, demonstrating the ability to determine the concentrations of the analytes. This work offers significant insight into developing novel SERS substrates, which is crucial for improving the detection limits of analytes.

One-dimensional scanning multiphoton imaging reveals prolonged calcium transient and sarcomere contraction in a zebrafish model of doxorubicin cardiotoxicity.

Doxorubicin (DOX) is a potent chemotherapeutic agent known to induce cardiotoxicity. Here we applied one-dimensional scanning multiphoton imaging to investigate the derangement of cardiac dynamics induced by DOX on a zebrafish model. DOX changed the cell morphology and significantly prolonged calcium transient and sarcomere contraction, leading to an arrhythmia-like contractile disorder. The restoration phase of calcium transient dominated the overall prolongation, indicating that DOX perturbed primarily the protein functions responsible for recycling cytosolic calcium ions. This novel finding supplements the existing mechanism of DOX cardiotoxicity. We anticipate that this approach should help mechanistic studies of drug-induced cardiotoxicity or heart diseases.

Selective induction of targeted cell death and elimination by near-infrared femtosecond laser ablation.

The techniques for inducing the death of specific cells in tissue has attracted attention as new methodologies for studying cell function and tissue regeneration. In this study, we show that a sequential process of targeted cell death and removal can be triggered by short-term exposure of near-infrared femtosecond laser pulses. Kinetic analysis of the intracellular accumulation of trypan blue and the assay of caspase activity revealed that femtosecond laser pulses induced immediate disturbance of plasma membrane integrity followed by apoptosis-like cell death. Yet, adjacent cells showed no sign of membrane damage and no increased caspase activity. The laser-exposed cells eventually detached from the substrate after a delay of >54 min while adjacent cells remained intact. On the base of in vitro experiments, we applied the same approach to ablate targeted single cardiac cells of a live zebrafish heart. The ability of inducing targeted cell death with femtosecond laser pulses should find broad applications that benefit from precise cellular manipulation at the level of single cells in vivo and in vitro.

Light triggering goldsomes enable local NO-generation and alleviate pathological vasoconstriction.

While nitric oxide (NO) can remedy vasoconstriction, inhalation of NO may cause systematic toxicity. We report a goldsome, which comprises a hollowed poly(lactic-co-glycolic acid) (PLGA) polymersome with S-nitrosoglutathione (GSNO, a NO donor) molecules and gold nanoparticles (Au NPs) incorporated in its hydrophilic core and hydrophobic membrane, respectively. Photothermal heating caused breakdown of polymersomes and enabled NO generation through reaction between GSNO and Au NPs. Photo-illumination at the zebrafish head led to local NO generation and selective cerebral vasodilation while it had little effects in regions away from the illumination site, and effectively mitigated hypoxia induced cerebral vasoconstriction. We demonstrate a translational potential by showing photo-stimulated NO generation with a clinical intravascular optical catheter. In conclusion, the goldsome, which enables light stimulated local NO generation and can be delivered with clinical intravascular optical catheters, should extend applications of NO therapies while surmounting limitations associated with systemic administration.

Reproducible and Bendable SERS Substrates with Tailored Wettability Using Block Copolymers and Anodic Aluminum Oxide Templates.

Surface properties are essential for substrates exhibiting high sensitivity in surface-enhanced Raman scattering (SERS) applications. In this work, novel SERS hybrid substrates using polystyrene-block-poly(methyl methacrylate) and anodic aluminum oxide templates is presented. The hybrid substrates not only possess hierarchical porous nanostructures but also exhibit superhydrophilic surface properties with the water contact angle ≈0°. Such surfaces play an important role in providing uniform enhanced intensities over large areas (relative standard deviation ≈10%); moreover, these substrates are found to be highly sensitive (limit of detection ≈10-12 m for rhodamine 6G (R6G)). The results show that the hybrid SERS substrates can achieve the simultaneous detection of multicomponent mixtures of different target molecules, such as R6G, crystal violet, and methylene blue. Furthermore, the bending experiments show that about 70% of the SERS intensities are maintained after bending from ≈30° to 150°.

Longitudinal and quantitative assessment platform for concurrent analysis of anti-tumor efficacy and cardiotoxicity of nano-formulated medication in vivo.

Increasing nanomedicinal approaches have been developed to effectively inhibit tumor growth; however, critical questions such as whether a nanomedicinal approach can mitigate latent side effects are barely addressed. To this end, we established a zebrafish xenograft tumor model, combining pseudodynamic three-dimensional cardiac imaging and image analysis to enable simultaneous and quantitative determination of the change of tumor volume and cardiac function of zebrafish upon specific nanoformulation treatment. Doxorubicin (DOX), a well-known chemotherapeutic agent with cardiotoxicity, and a recently developed DOX-loaded nanocomposite were employed as two model drugs to demonstrate the effectiveness to utilize the proposed evaluation platform for rapid validation. The nanoformulation significantly mitigated DOX-associated cardiotoxicity, while retaining the efficacy of DOX in inhibiting tumor growth compared to administration of carrier-free DOX at the same dose. We anticipate that this platform possesses the potential as an efficient assessment system for nanoformulated cancer therapeutics with suspected toxicity and side effects to vital organs such as the heart.

Zebrafish VCAP1X2 regulates cardiac contractility and proliferation of cardiomyocytes and epicardial cells.

Sarcomeric signaling complexes are important to sustain proper sarcomere structure and function, however, the mechanisms underlying these processes are not fully elucidated. In a gene trap experiment, we found that vascular cell adhesion protein 1 isoform X2 (VCAP1X2) mutant embryos displayed a dilated cardiomyopathy phenotype, including reduced cardiac contractility, enlarged ventricular chamber and thinned ventricular compact layer. Cardiomyocyte and epicardial cell proliferation was decreased in the mutant heart ventricle, as was the expression of pAKT and pERK. Contractile dysfunction in the mutant was caused by sarcomeric disorganization, including sparse myofilament, blurred Z-disc, and decreased gene expression for sarcomere modulators (smyd1b, mypn and fhl2a), sarcomeric proteins (myh6, myh7, vmhcl and tnnt2a) and calcium regulators (ryr2b and slc8a1a). Treatment of PI3K activator restored Z-disc alignment while injection of smyd1b mRNA restored Z-disc alignment, contractile function and cardiomyocyte proliferation in ventricles of VCAP1X2 mutant embryos. Furthermore, injection of VCAP1X2 variant mRNA rescued all phenotypes, so long as two cytosolic tyrosines were left intact. Our results reveal two tyrosine residues located in the VCAP1X2 cytoplasmic domain are essential to regulate cardiac contractility and the proliferation of ventricular cardiomyocytes and epicardial cells through modulating pAKT and pERK expression levels.

Controllable NO release from Cu1.6S nanoparticle decomposition of S-nitrosoglutathiones following photothermal disintegration of polymersomes to elicit cerebral vasodilatory activity.

Since the discovery of nitric oxide (NO) as a vasodilator, numerous NO therapies have been attempted to remedy disorders related to pathological vasoconstriction such as coronary artery disease. Despite the advances, clinical applications of NO therapies remain limited mainly because of the low stability of molecular NO donors (and NO molecules), and concerns about the increased oxidative stress and reduced arterial pressure associated with the systemic administration of NO. Here we design a photo-responsive polymersome with nitrosothiols and Cu1.6S nanoparticles in its core and shell, respectively, and demonstrate the photo-triggered release of NO and its vasodilatory activity on zebrafish. Unlike conventional approaches, our design enhances the stability of NO donors and prospectively enables spatiotemporal regulation of NO release, thus minimizing the harmful effects associated with conventional NO therapies. We anticipate that such a strategy will open up new clinical applications of NO and help reveal the complex biological effects of NO in vivo.

Zebrafish model of photochemical thrombosis for translational research and thrombolytic screening in vivo.

Acute thromboembolic diseases remain the major global cause of death or disability. Although an array of thrombolytic and antithrombotic drugs has been approved to treat or prevent thromboembolic diseases, many more drugs that target specific clotting mechanisms are under development. Here a novel zebrafish model of photochemical thrombosis is reported and its prospective application for the screening and preclinical testing of thrombolytic agents in vivo is demonstrated. Through photochemical excitation, a thrombus was induced to form at a selected section of the dorsal aorta of larval zebrafish, which had been injected with photosensitizers. Such photochemical thrombosis can be consistently controlled to occlude partially or completely the targeted blood vessel. Detailed mechanistic tests indicate that the zebrafish model of photochemical thrombosis exhibits essential features of classical coagulation and a thrombolytic pathway. For demonstration, tissue plasminogen activator (tPA), a clinically feasible thrombolytic agent, was shown to effectively dissolve photochemically induced blood clots. In light of the numerous unique advantages of zebrafish as a model organism, our approach is expected to benefit not only the development of novel thrombolytic and antithrombotic strategies but also the fundamental or translational research targeting hereditary thrombotic or coagulation disorders.

Toward live-cell imaging of dopamine neurotransmission with fluorescent neurotransmitter analogues.

We report a novel 'fluorescent dopamine' that possesses essential features of natural dopamine. Our method is simple and is readily extended to monoamine neurotransmitters such as L-norepinephrine, serotonin and GABA, providing a more practical approach. Because of its compatibility with sensitive fluorescent measurements, we envisage that our approach will have a broad range of applications in neural research.

Multiphoton microscopy in defining liver function.

Multiphoton microscopy is the preferred method when in vivo deep-tissue imaging is required. This review presents the application of multiphoton microscopy in defining liver function. In particular, multiphoton microscopy is useful in imaging intracellular events, such as mitochondrial depolarization and cellular metabolism in terms of NAD(P)H changes with fluorescence lifetime imaging microscopy. The morphology of hepatocytes can be visualized without exogenously administered fluorescent dyes by utilizing their autofluorescence and second harmonic generation signal of collagen, which is useful in diagnosing liver disease. More specific imaging, such as studying drug transport in normal and diseased livers are achievable, but require exogenously administered fluorescent dyes. If these techniques can be translated into clinical use to assess liver function, it would greatly improve early diagnosis of organ viability, fibrosis, and cancer.

Optical assessment of the cardiac rhythm of contracting cardiomyocytes in vitro and a pulsating heart in vivo for pharmacological screening.

Our quest in the pathogenesis and therapies targeting human heart diseases requires assessment of the contractile dynamics of cardiac models of varied complexity, such as isolated cardiomyocytes and the heart of a model animal. It is hence beneficial to have an integral means that can interrogate both cardiomyocytes in vitro and a heart in vivo. Herein we report an application of dual-beam optical reflectometry to determine noninvasively the rhythm of two representative cardiac models-chick embryonic cardiomyocytes and the heart of zebrafish. We probed self-beating cardiomyocytes and revealed the temporally varying contractile frequency with a short-time Fourier transform. Our unique dual-beam setup uniquely records the atrial and ventricular pulsations of zebrafish simultaneously. To minimize the cross talk between signals associated with atrial and ventricular chambers, we particularly modulated the two probe beams at distinct frequencies and extracted the signals specific to individual cardiac chambers with phase-sensitive detection. With this setup, we determined the atrio-ventricular interval, a parameter that is manifested by the electrical conduction from the atrium to the ventricle. To demonstrate pharmacological applications, we characterized zebrafish treated with various cardioactive and cardiotoxic drugs, and identified abnormal cardiac rhythms and atrioventricular (AV) blocks of varied degree. In light of its potential capability to assess cardiac models both in vitro and in vivo and to screen drugs with cardioactivity or toxicity, we expect this approach to have broad applications ranging from cardiopharmacology to developmental biology.

Molecular imaging of ischemia and reperfusion in vivo with mitochondrial autofluorescence.

Ischemia and reperfusion (IR) injury constitutes a pivotal mechanism of tissue damage in pathological conditions such as stroke, myocardial infarction, vascular surgery, and organ transplant. Imaging or monitoring of the change of an organ at a molecular level in real time during IR is essential to improve our understanding of the underlying pathophysiology and to guide therapeutic strategies. Herein, we report molecular imaging of a rat model of hepatic IR with the autofluorescence of mitochondrial flavins. We demonstrate a revelation of the histological characteristics of a liver in vivo with no exogenous stain and show that intravital autofluorescent images exhibited a distinctive spatiotemporal variation during IR. The autofluorescence decayed rapidly from the baseline immediately after 20-min ischemia (approximately 30% decrease in 5 min) but recovered gradually during reperfusion (to approximately 99% of the baseline 9 min after the onset of reperfusion). The autofluorescent images acquired during reperfusion correlated strongly with the reperfused blood flow. We show further that the autofluorescence was produced predominantly from mitochondria, and the distinctive autofluorescent variation during IR was mechanically linked to the altered balance between the flavins in the oxidized and reduced forms residing in the mitochondrial electron-transport chain. Our approach opens an unprecedented route to interrogate the deoxygenation and reoxygenation of mitochondria, the machinery central to the pathophysiology of IR injury, with great molecular specificity and spatiotemporal resolution and can be prospectively translated into a medical device capable of molecular imaging. We envisage that the realization thereof should shed new light on clinical diagnostics and therapeutic interventions targeting IR injuries of not only the liver but also other vital organs including the brain and heart.

Characterization of the pharmaceutical effect of drugs on atherosclerotic lesions in vivo using integrated fluorescence imaging and Raman spectral measurements.

Direct assessment of the vascular lesions of model animals in vivo is important for the development of new antiatherosclerotic drugs. Nevertheless, biochemical analysis of the lipid profile in blood in vitro remains the most common way to evaluate the therapeutic effect of drugs targeting atherosclerosis because of an inherent difficulty to access the vascular wall. Using hypercholesterolemic zebrafish, we present an orchestrated application of Raman spectral measurements and confocal fluorescence imaging to interrogate the pharmacological response of atherosclerotic lesions in situ and in vivo. For demonstration, we investigated two commonly prescribed antihyperlipidemic drugs, ezetimibe and atorvastatin. The treatment of ezetimibe or atorvastatin alone decreased effectively the deposition of lipids in the vascular wall, and a combined dose showed a synergistic effect. Atorvastatin exerted a profound antioxidative effect on vascular fatty lesions. Analysis of individual lesions shows further that these lesions exhibited a heterogeneous response to the treatment of atorvastatin; a significant fraction of, but not all, the lesions became nonoxidized after the intervention. Beyond its efficacies in suppressing both the accumulation and oxidation of vascular lipids, atorvastatin expedited the clearance of vascular lipids. The possession of pleotropic (multiple) therapeutic effects on vascular fatty lesions of hypercholesterolemic zebrafish by atorvastatin is notably consistent with the known pharmaceutical effects of this drug on human beings. These results improve our understanding of the antiatherosclerotic effect of drugs. We envisage that our approach has the potential to become a platform to predict the pharmaceutical effects of new drugs aiming to cure human atherosclerotic diseases.

Toward functional screening of cardioactive and cardiotoxic drugs with zebrafish in vivo using pseudodynamic three-dimensional imaging.

Given the high mortality in patients with cardiovascular diseases and the life-threatening consequences of drugs with unforeseen adverse effects on hearts, a critical evaluation of the pharmacological response of cardiovascular function on model animals is important especially in the early stages of drug development. We report a proof-of-principle study to demonstrate the utility of zebrafish as an analytical platform to predict the cardiac response of new drugs or chemicals on human beings. With pseudodynamic 3D imaging, we derive individual parameters that are central to the cardiac function of zebrafish, including the ventricular stroke volume, ejection fraction, cardiac output, heart rate, diastolic filling function, and ventricular mass. We evaluate both inotropic and chronotropic responses of the heart of zebrafish treated with drugs that are commonly prescribed and possess varied known cardiac activities. We reveal deranged cardiac function of a zebrafish model of cardiomyopathy induced with a cardiotoxic drug. The cardiac function of zebrafish exhibits a pharmacological response similar to that of human beings. We compare also cardiac parameters obtained in this work with those derived with conventional 2D approximation and show that the latter tends to overestimate the cardiac parameters and produces results of greater variation. In view of the growing interest of using zebrafish in both fundamental and translational biomedical research, we envisage that our approach should benefit not only contemporary pharmaceutical development but also exploratory research such as gene, stem cell, or regenerative therapies targeting congenital or acquired heart diseases.

Oligonucleotides as 'bio-solvent' for in situ extraction and functionalisation of carbon nanoparticles.

A simple yet novel one-pot approach is developed to prepare carbon nanoparticles with diameters of ∼2 nm and modified by oligonucleotides. We use single-stranded deoxyribonucleic acid (ssDNA), which serves as a unique 'bio-solvent' for carbon nanoparticle (CNP) preparation and as a target molecule for functionalisation. Proposed interactions relevant to the stabilisation of the final oligonucleotide-CNP complex include π-π stacking and π-HN bonding with sp2 carbon atoms on the CNP surface. Furthermore, oligonucleotide-enriched CNPs can be readily extracted within seconds from a crude mixture of single-walled carbon nanotubes (SWCNTs) without any need for post-synthesis chemical modification. The established CNPs are biocompatible, possess intrinsic fluorescence, and do not result in the undesirable photobleaching effect, rendering them potential candidates for in vivo biological applications.

Characterization of the mechanodynamic response of cardiomyocytes with atomic force microscopy.

Coordinated and synchronous contraction of cardiomyocytes ensures a normal cardiac function while deranged contraction of cardiomyocytes can lead to heart failure and circulatory dysfunction. Detailed assessment of the contractile property of cardiomyocytes not only helps elucidate the pathophysiology of heart failure but also facilitates development of novel therapies. Herein, we report application of atomic force microscopy to determine essential mechanodynamic characteristics of self-beating cardiomyocytes including the contractile amplitude, force, and frequency. The contraction was continuously measured on the same point of the cell surface; the result assessed postintervention was then compared with the baseline, and the fractional change was obtained. We employed short-time Fourier transform to analyze the time-varying contractile properties and calculate the spectrogram, based on which subtle dynamic changes in the contractile rhythmicity were delicately illustrated. To demonstrate potential applications of this approach, we examined the inotropic and chronotropic responses of cardiomyocyte contraction induced by various pharmacological interventions. The administration of epinephrine significantly increased the contractile amplitude, force, and frequency whereas esmolol markedly decreased these contractile properties. As uniquely illustrated in the spectrogram, doxorubicin not only impaired the contractility of cardiomyocytes but also drastically compromised the rhythmicity. We envision that our approach should be useful in research fields that require detailed evaluation of the mechanodynamic response of cardiomyocytes, for example, to screen drugs that possess cardiac activity or cardiotoxicity, or to assess chemicals that could direct differentiation of stem cells into functioning cardiomyocytes.

Imaging of zebrafish in vivo with second-harmonic generation reveals shortened sarcomeres associated with myopathy induced by statin.

We employed second-harmonic generation (SHG) imaging and the zebrafish model to investigate the myopathy caused by statin in vivo with emphasis on the altered microstructures of the muscle sarcomere, the fundamental contractile element of muscles. This approach derives an advantage of SHG imaging to observe the striated skeletal muscle of living zebrafish based on signals produced mainly from the thick myosin filament of sarcomeres without employing exogenous labels, and eliminates concern about the distortion of muscle structures caused by sample preparation in conventional histological examination. The treatment with statin caused a significantly shortened sarcomere relative to an untreated control (1.73±0.09 µm vs 1.91±0.08 µm, P<0.05) while the morphological integrity of the muscle fibers remained largely intact. Mechanistic tests indicated that this microstructural disorder was associated with the biosynthetic pathway of cholesterol, or, specifically, with the impaired production of mevalonate by statins. This microstructural disorder exhibited a strong dependence on both the dosage and the duration of treatment, indicating a possibility to assess the severity of muscle injury according to the altered length of the sarcomeres. In contrast to a conventional assessment of muscle injury using clinical biomarkers in blood, such as creatine kinase that is released from only disrupted myocytes, the ability to determine microstructural modification of sarcomeres allows diagnosis of muscle injury before an onset of conventional clinical symptoms. In light of the increasing prevalence of the incidence of muscle injuries caused by new therapies, our work consolidates the combined use of the zebrafish and SHG imaging as an effective and sensitive means to evaluate the safety profile of new therapeutic targets in vivo.

Selective and absolute quantification of endogenous hypochlorous acid with quantum-dot conjugated microbeads.

Endogenous hypochlorous acid (HOCl) secreted by leukocytes plays a critical role in both the immune defense of mammalians and the pathogenesis of various diseases intimately related to inflammation. We report the first selective and absolute quantification of endogenous HOCl produced by leukocytes in vitro and in vivo with a novel quantum dot-based sensor. An activated human neutrophil secreted 6.5 ± 0.9 × 10(8) HOCl molecules into its phagosome, and kinetic measurement for the secretions showed that the extracellular generation of HOCl was temporally retarded, but the quantity eventually attained a level comparable with its intraphagosomal counterpart with a delay of about 1.5 h. The quantity of HOCl secreted from the hepatic leukocytes of rats with or without stimulation of lipopolysaccharide was also determined. These results indicate a possibility to extend our approach to not only clinical settings for quantitative assessment of the bactericidal capability of isolated leukocytes of patients but also fundamental biomedical research that requires critical evaluation of the inflammatory response of animals.

CNS-targeted AAV5 gene transfer results in global dispersal of vector and prevention of morphological and function deterioration in CNS of globoid cell leukodystrophy mouse model.

Globoid cell leukodystrophy (GLD) is a devastating lysosomal storage disease caused by deficiency of the enzyme galactocerebrosidase (GALC). Currently, there is no definite cure for GLD. Several attempts with CNS-directed gene therapy in twitcher mice (a murine model of GLD) demonstrated restricted expression of GALC activity in CNS and failure of therapeutic efficacy in cerebellum and spinal cord, resulting in various degrees of correction of biochemical, pathological and clinical phenotype. More recently, twitcher mice receiving a combination of hematopoietic and viral vector gene transfer therapies were not protected from neurodegeneration and axonopathy in both cerebellum and spinal cord. This evidence indicates the requirement of sufficient and widespread GALC expression in CNS and rescue of cerebellum and spinal cord in the therapeutic intervention of murine model of GLD. In this study, we have optimized intracranial delivery of AAV2/5-GALC to the neocortex, hippocampus and cerebellum, instead of the thalamus as was previously conducted, of twitcher mice. The CNS-targeted AAV2/5 gene transfer effectively dispersed GALC transgene along the neuraxis of CNS as far as the lumbar spinal cord, and reduced the accumulation of psychosine in the CNS of twitcher mice. Most importantly, the treated twitcher mice were protected from loss of oligodendrocytes and Purkinje cells, axonopathy and marked gliosis, and had significantly improved neuromotor function and prolonged lifespan. These preclinical findings with our approach are encouraging, although a more robust response in the spinal cord would be desirable. Collectively, the information in this study validates the efficacy of this gene delivery approach to correct enzymatic deficiency, psychosine accumulation and neuropathy in CNS of GLD. Combining cell therapy such as bone marrow transplantation with treatment with the aim of reducing inflammation, replacing dead or dying oligodendrocytes and targeting PNS may provide a synergistic and more complete correction of this disease.