scholarly journals Real-time in vivo imaging of size-dependent transport and toxicity of gold nanoparticles in zebrafish embryos using single nanoparticle plasmonic spectroscopy

2013 ◽  
Vol 3 (3) ◽  
pp. 20120098 ◽  
Author(s):  
Lauren M. Browning ◽  
Tao Huang ◽  
Xiao-Hong Nancy Xu
Keyword(s):  
Embryonic Development ◽  
Real Time ◽  
Rational Design ◽  
Dark Field ◽  
Molecular Probes ◽  
Noble Metal Nanoparticles ◽  
Zebrafish Embryos ◽  
Ag Nps ◽  
Au Nps

Noble metal nanoparticles (NPs) show distinctive plasmonic optical properties and superior photostability, enabling them to serve as photostable multi-coloured optical molecular probes and sensors for real-time in vivo imaging. To effectively study biological functions in vivo , it is essential that the NP probes are biocompatible and can be delivered into living organisms non-invasively. In this study, we have synthesized, purified and characterized stable (non-aggregated) gold (Au) NPs (86.2 ± 10.8 nm). We have developed dark-field single NP plasmonic microscopy and spectroscopy to study their transport into early developing zebrafish embryos (cleavage stage) and their effects on embryonic development in real-time at single NP resolution. We found that single Au NPs (75–97 nm) passively diffused into the embryos via their chorionic pore canals, and stayed inside the embryos throughout their entire development (120 h). The majority of embryos (96 ± 3%) that were chronically incubated with the Au NPs (0–20 pM) for 120 h developed to normal zebrafish, while an insignificant percentage of embryos developed to deformed zebrafish (1 ± 1)% or dead (3 ± 3)%. Interestingly, we did not observe dose-dependent effects of the Au NPs (0–20 pM) on embryonic development. By comparing with our previous studies of smaller Au NPs (11.6 ± 0.9 nm) and similar-sized Ag NPs (95.4 ± 16.0 nm), we found that the larger Au NPs are more biocompatible than the smaller Au NPs, while the similar-sized Ag NPs are much more toxic than Au NPs. This study offers in vivo assays and single NP microscopy and spectroscopy to characterize the biocompatibility and toxicity of single NPs, and new insights into the rational design of more biocompatible plasmonic NP imaging probes.

scholarly journals Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yusaku Hontani ◽  
Mikhail Baloban ◽  
Francisco Velazquez Escobar ◽  
Swetta A. Jansen ◽  
Daria M. Shcherbakova ◽  
...  
Keyword(s):  
Real Time ◽  
Rational Design ◽  
Near Infrared ◽  
Fluorescent Proteins ◽  
Covalent Bond ◽  
Binding Pocket ◽  
Tissue Imaging ◽  
Covalent Attachment ◽  
Time Resolved

AbstractNear-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C–S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

scholarly journals Marine Bacterial Exopolymers-Mediated Green Synthesis of Noble Metal Nanoparticles with Antimicrobial Properties

Polymers ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 1157 ◽  
Author(s):  
Angela Scala ◽  
Anna Piperno ◽  
Alexandru Hada ◽  
Simion Astilean ◽  
Adriana Vulpoi ◽  
...  
Keyword(s):  
Hydrothermal Vents ◽  
Thermophilic Bacteria ◽  
Silver Chloride ◽  
Antimicrobial Properties ◽  
Biological Evaluation ◽  
Noble Metal Nanoparticles ◽  
Ag Nps ◽  
Au Nps ◽  
Minimum Fungicidal Concentration ◽  
Gold Silver

A straightforward and green method for the synthesis of gold, silver, and silver chloride nanoparticles (Au NPs and Ag/AgCl NPs) was developed using three different microbial exopolymers (EP) as reducing and stabilizing agents. The exopolysaccharides EPS B3-15 and EPS T14 and the poly-γ-glutamic acid γ-PGA-APA were produced by thermophilic bacteria isolated from shallow hydrothermal vents off the Eolian Islands (Italy) in the Mediterranean Sea. The production of metal NPs was monitored by UV−Vis measurements by the typical plasmon resonance absorption peak and their antimicrobial activity towards Gram-positive and Gram- negative bacteria (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa), as well as fungi (Candida albicans) was investigated. The biological evaluation showed no activity for EP-Au NPs, except against E. coli, whereas EP-Ag NPs exhibited a broad-spectrum of activity. The chemical composition, morphology, and size of EP-Ag NPs were investigated by UV–Vis, zeta potential (ζ), dynamic light scattering (DLS) measurements and transmission electron microscopy (TEM). The best antimicrobial results were obtained for EPS B3-15-Ag NPs and EPS T14-Ag NPs (Minimum Inhibitory Concentration, MIC: 9.37–45 µg/mL; Minimum Bactericidal Concentration/Minimum Fungicidal Concentration, MBC/MFC: 11.25–75 µg/mL).

scholarly journals Real-time Ratiometric Imaging of Micelles Assembly State in a Microfluidic Cancer-on-a-chip

2020 ◽  
Author(s):  
Natalia Feiner-Gracia ◽  
Adrianna Glinkowska Mares ◽  
Marina Buzhor ◽  
Romen Rodriguez-Trujillo ◽  
Josep Samitier ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Drug Release ◽  
Real Time ◽  
Rational Design ◽  
Confocal Imaging ◽  
Fluorescent Properties ◽  
Key Factor ◽  
Low Efficiency

ABSTRACTThe performance of supramolecular nanocarriers as drug delivery systems depends on their stability in the complex and dynamic biological media. After administration, nanocarriers are challenged by confronting different barriers such as shear stress and proteins present in blood, endothelial wall, extracellular matrix and eventually cancer cell membranes. While early disassembly will result in a premature drug release, extreme stability of the nanocarriers can lead to poor drug release and low efficiency. Therefore, comprehensive understanding of the stability and assembly state of supramolecular carriers in each stage of delivery is a key factor for the rational design of these systems. One of the key challenges is that current 2D in vitro models do not provide exhaustive information, as they do not fully recapitulate the 3D tumor microenvironment. This deficiency of the 2D models complexity is the main reason for the differences observed in vivo when testing the performance of supramolecular nanocarriers. Herein, we present a real-time monitoring study of self-assembled micelles stability and extravasation, combining spectral confocal microscopy and a microfluidic tumor-on-a-chip. The combination of advanced imaging and a reliable organ-on-a-chip model allow us to track micelle disassembly by following the spectral properties of the amphiphiles in space and time during the crucial steps of drug delivery. The spectrally active micelles were introduced under flow and their position and conformation followed during the crossing of barriers by spectral imaging, revealing the interplay between carrier structure, micellar stability and extravasation. Integrating the ability of the micelles to change their fluorescent properties when disassembled, spectral confocal imaging and 3D microfluidic tumor blood vessel-on-a-chip, resulted in the establishment of a robust testing platform, suitable for real-time imaging and evaluation of supramolecular drug delivery carrier’s stability.

scholarly journals In Vivo Metabolic Response upon Exposure to Gold Nanorod Core/Silver Shell Nanostructures: Modulation of Inflammation and Upregulation of Dopamine

2020 ◽  
Vol 21 (2) ◽  
pp. 384 ◽  
Author(s):  
Haiyun Li ◽  
Tao Wen ◽  
Tao Wang ◽  
Yinglu Ji ◽  
Yaoyi Shen ◽  
...  
Keyword(s):  
Rational Design ◽  
Molecular Mechanisms ◽  
Metabolic Response ◽  
Dopamine Metabolism ◽  
Gold Nanorod ◽  
Ag Nps ◽  
Shell Nanostructures ◽  
Antioxidant Defense Systems ◽  
Silver Shell

With the increasing applications of silver nanoparticles (Ag NPs), the concerns of widespread human exposure as well as subsequent health risks have been continuously growing. The acute and chronic toxicities of Ag NPs in cellular tests and animal tests have been widely investigated. Accumulating evidence shows that Ag NPs can induce inflammation, yet the overall mechanism is incomplete. Herein, using gold nanorod core/silver shell nanostructures (Au@Ag NRs) as a model system, we studied the influence on mice liver and lungs from the viewpoint of metabolism. In agreement with previous studies, Au@Ag NRs’ intravenous exposure caused inflammatory reaction, accompanying with metabolic alterations, including energy metabolism, membrane/choline metabolism, redox metabolism, and purine metabolism, the disturbances of which contribute to inflammation. At the same time, dopamine metabolism in liver was also changed. This is the first time to observe the production of dopamine in non-neural tissue after treatment with Ag NPs. As the upregulation of dopamine resists inflammation, it indicates the activation of antioxidant defense systems against oxidative stress induced by Au@Ag NRs. In the end, our findings deepened the understanding of molecular mechanisms of Ag NPs-induced inflammation and provide assistance in the rational design of their biomedical applications.

Doping Concentration Tuning and Plasmonic Optical Properties Modelling of Metal Nano Particles Utilizing FDTD Method

2020 ◽  
Vol 995 ◽  
pp. 197-202
Author(s):  
Mehran Rafiee ◽  
Subhash Chandra ◽  
Hind Ahmed ◽  
Aaron Glenn ◽  
Conor Mc Loughlin ◽  
...  
Keyword(s):  
Optical Properties ◽  
Doping Concentration ◽  
Radiative Decay ◽  
Fdtd Method ◽  
Nano Particles ◽  
Noble Metal Nanoparticles ◽  
Field Energy ◽  
Ag Nps ◽  
Gold And Silver Nanoparticles ◽  
Au Nps

Among noble metal nanoparticles (MNPs), plasmonic effect of gold and silver nanoparticles (Au NPs and Ag NPs) is interesting to study due to their high near-field energy. This makes them excellent particles to absorb and scatter incident radiation in optical applications such as coupling them to fluorescent emitters to enhance their radiative decay and emission rate. This paper reviews and studies the methodology required for tuning the doping concentration and modelling plasmonic optical properties of Au NPs and Ag NPs by using finite difference time domain (FDTD) method. The frequency-dependent optical behaviour of MNPs is discussed. Plasmonic optical properties of MNPs can be characterised by their dimension, shape and doping concentration. Dimension and shape of MNPs can be simply defined in FDTD space grid (known as Yee grid). However, tuning MNPs doping concentration is more challenging which is undertaken by changing the spacing between MNPs and lateral boundaries of FDTD Yee grid. This can be vastly useful in Au NPs and Ag NPs optical optimization and applications to estimate plasmonic resonance based on doping concentration of particles.

scholarly journals Cryptolepine, the Main Alkaloid of the Antimalarial Cryptolepis sanguinolenta (Lindl.) Schlechter, Induces Malformations in Zebrafish Embryos

2019 ◽  
Vol 2019 ◽  
pp. 1-7 ◽  
Author(s):  
Kwesi Boadu Mensah ◽  
Charles Benneh ◽  
Arnold Donkor Forkuo ◽  
Charles Ansah
Keyword(s):  
Embryonic Development ◽  
Ex Vivo ◽  
Sensitive Period ◽  
Mercury Chloride ◽  
Hatching Rate ◽  
Zebrafish Embryos ◽  
Benzyl Penicillin ◽  
Cryptolepis Sanguinolenta ◽  
Embryo Lethality

Background. Previous studies on cryptolepine, the antimalarial and cytotoxic alkaloid of Cryptolepis sanguinolenta, showed that it preferentially accumulates in rapidly proliferating cells and melanin-containing tissues. Subsequently, we demonstrated that cryptolepine was toxic to murine embryos in vivo but no signs of teratogenicity. in vivo developmental studies can be confounded by maternal effects. Here, we hypothesized that cryptolepine-induced embryo toxicity occurs at least partly through direct inhibition of embryogenesis rather than indirectly through the induction of maternal toxicity. Aim. To determine the effects of cryptolepine on developing zebrafish embryos ex vivo. Methods. Healthy synchronized zebrafish eggs were treated with cryptolepine (10−1 − 5 × 102 μM), benzyl penicillin (6 − 6 × 102 μM), or mercury chloride (3.7 × 10−1 − 3.7 × 101 nM) from 6 to 72 hours postfertilization. Developing embryos were assessed at 24, 48, 72, and 96 hours under microscope for lethality, hatching rate, and malformation. Results. LC50 for cryptolepine in the study was found to be 260 ± 0.174 μM. Cryptolepine induced dose- and time-dependent mortality from the 24 to 96 hours postfertilization. Lower cryptolepine concentration (<100 μM) caused mortality, approximately 15–18%, only after the 48 hours postfertilization. The most sensitive period of embryo lethality corresponded well with the pharyngula (24 to 48 hours) and hatching (48 to 72 hours) stages of embryonic development. Cryptolepine (10−1 − 5 × 102 μM) dose dependently inhibited the hatching rate. At doses above 500 μM, hatching was completely inhibited. Mercury chloride (3.7 × 10−1 − 3.7 × 101 nM), used as positive control, induced a consistent pattern of embryo lethality at all stages of development, whereas benzyl penicillin (6 − 6 × 102 μM), used as negative control, did not induce any significant embryo lethality. Morphological examination of (postfertilization day 5) of eleutheroembryos treated during embryonic development with cryptolepine showed decreased body length (growth inhibition), decreased eye diameter and bulginess, enlarged pericardia, and enlarged yolk sac and muscle malformations. Conclusion. Cryptolepine induces malformations, growth retardation, and mortalities in rapidly dividing zebrafish embryos ex vivo.

scholarly journals Extracellular Vesicle-delivered Bone Morphogenetic Proteins: A novel paracrine mechanism during embryonic development

2018 ◽  
Author(s):  
Thomas Draebing ◽  
Jana Heigwer ◽  
Lonny Juergensen ◽  
Hugo Albert Katus ◽  
David Hassel
Keyword(s):  
Embryonic Development ◽  
Bone Morphogenetic Proteins ◽  
Extracellular Vesicle ◽  
Biologically Active ◽  
Zebrafish Embryos ◽  
Bmp Signalling ◽  
Endodermal Cells ◽  
Morphogen Transport

Morphogens including Wnt, Hedgehog and BMP proteins are essential during embryonic development and early induction of organ progenitors. Besides free diffusion to form signalling gradients, extracellular vesicle- (EV-) mediated morphogen transport was identified as a central mechanism for Wnt- and Hh-signalling. Here, we investigated EVs isolated from whole zebrafish embryos as a potential morphogen transport mechanism. Inhibition of EV-secretion during development leads to severe dorsalization phenotypes, reminiscent of disrupted BMP-signalling. Subsequently, we found that EVs isolated from zebrafish embryos at bud stage contain biologically active BMP2/4 protein. Embryos with inhibited EV secretion display reduced Smad1/5/9-phosphorylation and downstream gene expression activity. We further show that BMP-containing EVs are secreted by endodermal cells in vitro, and inhibition of endodermal-EV release in vivo causes signs of BMP signalling loss. Our data provides evidence that establishes the transport of BMP2/4 by EVs as an essential but so far undiscovered mechanism in developmental morphogenesis.

145 CHARACTERIZATION OF THE PROTEIN ARGININE METHYLTRANSFERASE-DIMETHYLARGININE DIMETHYLAMINOHYDROLASE-NITRIC OXIDE AXIS DURING PORCINE EMBRYO DEVELOPMENT

2012 ◽  
Vol 24 (1) ◽  
pp. 185
Author(s):  
K. Tessanne ◽  
B. Redel ◽  
K. Whitworth ◽  
L. Spate ◽  
A. Brown ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Embryonic Development ◽  
Real Time ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
No Production ◽  
Dimethylarginine Dimethylaminohydrolase ◽  
Porcine Embryo

Transcriptional deep sequencing analysis by Bauer et al. (2010) revealed a significant increase in expression of the arginine transporter SLC7A1 in in vitro–cultured porcine blastocysts compared with those cultured in vivo and this was corrected through supplemental arginine. This indicates an important role for arginine during porcine embryo development. Arginine is the precursor for nitric oxide (NO) production and previous work in mice and cattle has shown decreased development when embryos were cultured with a nitric oxide synthase (NOS) inhibitor. The NOS activity is inhibited by monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA) that are released during degradation of proteins methylated by protein arginine methyltransferases (PRMT). The enzyme dimethylarginine dimethylaminohydrolase (DDAH) is responsible for degrading MMA and ADMA in the cell. Therefore, the goal of this study was to investigate whether this PRMT-DDAH-NO axis exists in pre-implantation porcine embryos. To this end, expression of PRMT1, PRMT3, PRMT5, DDAH1 and endothelial NOS (NOS3) was analysed at different stages of embryonic development using real-time quantitative RT-PCR. In addition, the effect of supplemental arginine (1.69 mM) on the expression of the aforementioned genes was investigated. Production of NO in porcine embryos was also visualised using 4-amino-5-methylamino-2,7-difluorofluorescein diacetate (DAF-FM-DA). In vitro–fertilized porcine embryos were collected at the 4-cell and blastocyst stages. The RNA was isolated from pools of 18 to 20 embryos and cDNA, was synthesised using Superscript III (Invitrogen, Carlsbad, CA, USA). Real-time PCR analysis was performed and the mean fold change in gene expression from the reference gene YWHAG was analysed by t-test after a log transformation. Expression of PRMT3 and PRMT5 was significantly higher (P < 0.05) in blastocysts versus 4-cell embryos. Expression of PRMT1, however, was higher in 4-cell embryos (P < 0.05). The expression of DDAH1 was detected in 4-cell embryos, but DDAH1 became undetectable by the blastocyst stage. Previous microarray analysis in our laboratory by Whitworth et al. (2005 Biol. Reprod. 72(6), 1437–1451) also revealed a significant up-regulation of DDAH2 expression at the 4-cell stage versus blastocysts. Expression of NOS3 was undetectable in the 4-cell and blastocyst; however, NO was detected in 4-cell and blastocyst stage embryos by using DAF-FM-DA. This suggests that a different NOS may be acting in the porcine embryo. Addition of arginine did not have a significant effect on expression of the analysed genes. These results suggest that PRMT-DDAH regulated NO production may play a role during porcine embryo development. Understanding the PRMT-DDAH-NO axis and its regulation during embryonic development will further our ability to tailor in vitro culture so that it more appropriately mimics that of an in vivo environment. Funding was provided by NIH U42 RR18877.

scholarly journals An Adjustable Dark-Field Acoustic-Resolution Photoacoustic Imaging System with Fiber Bundle-Based Illumination

Biosensors ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 262
Author(s):  
Yuhling Wang ◽  
De-Fu Jhang ◽  
Tsung-Sheng Chu ◽  
Chia-Hui Tsao ◽  
Chia-Hua Tsai ◽  
...  
Keyword(s):  
Real Time ◽  
Imaging System ◽  
Structural Information ◽  
Photoacoustic Imaging ◽  
Biological Tissues ◽  
Dark Field ◽  
Design Concept ◽  
High Frequency Ultrasound

Photoacoustic (PA) imaging has become one of the major imaging methods because of its ability to record structural information and its high spatial resolution in biological tissues. Current commercialized PA imaging instruments are limited to varying degrees by their bulky size (i.e., the laser or scanning stage) or their use of complex optical components for light delivery. Here, we present a robust acoustic-resolution PA imaging system that consists of four adjustable optical fibers placed 90° apart around a 50 MHz high-frequency ultrasound (US) transducer. In the compact design concept of the PA probe, the relative illumination parameters (i.e., angles and fiber size) can be adjusted to fit different imaging applications in a single setting. Moreover, this design concept involves a user interface built in MATLAB. We first assessed the performance of our imaging system using in vitro phantom experiments. We further demonstrated the in vivo performance of the developed system in imaging (1) rat ear vasculature, (2) real-time cortical hemodynamic changes in the superior sagittal sinus (SSS) during left-forepaw electrical stimulation, and (3) real-time cerebral indocyanine green (ICG) dynamics in rats. Collectively, this alignment-free design concept of a compact PA probe without bulky optical lens systems is intended to satisfy the diverse needs in preclinical PA imaging studies.

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