scholarly journals Integrin αvβ6-targeted MR molecular imaging of breast cancer in a xenograft mouse model

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Dengfeng Li ◽  
Chengyan Dong ◽  
Xiaohong Ma ◽  
Xinming Zhao
Keyword(s):  
Breast Cancer ◽  
Molecular Imaging ◽  
Magnetic Resonance ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Hek293 Cells ◽  
Targeted Nanoparticles ◽  
Scanning Confocal Microscopy

Abstract Background The motif RXDLXXL-based nanoprobes allow specific imaging of integrin αvβ6, a protein overexpressed during tumorigenesis and tumor progression of various tumors. We applied a novel RXDLXXL-coupled cyclic arginine-glycine-aspartate (RGD) nonapeptide conjugated with ultrasmall superparamagnetic iron oxide nanoparticles (referred to as cFK-9-USPIO) for the application of integrin αvβ6-targeted magnetic resonance (MR) molecular imaging for breast cancer. Methods A novel MR-targeted nanoprobe, cFK-9-USPIO, was synthesized by conjugating integrin αvβ6-targeted peptide cFK-9 to N-amino (−NH2)-modified USPIO nanoparticles via a dehydration esterification reaction. Integrin αvβ6-positive mouse breast cancer (4 T1) and integrin αvβ6 negative human embryonic kidney 293 (HEK293) cell lines were incubated with cFK-9-AbFlour 647 (blocking group) or cFK-9-USPIO (experimental group), and subsequently imaged using laser scanning confocal microscopy (LSCM) and 3.0 Tesla magnetic resonance imaging (MRI) system. The affinity of cFK-9 targeting αvβ6 was analyzed by calculating the mean fluorescent intensity in cells, and the nanoparticle targeting effect was measured by the reduction of T2 values in an in vitro MRI. The in vivo MRI capability of cFK-9-USPIO was investigated in 4 T1 xenograft mouse models. Binding of the targeted nanoparticles to αvβ6-positive 4 T1 tumors was determined by ex vivo histopathology. Results In vitro laser scanning confocal microscopy (LSCM) imaging showed that the difference in fluorescence intensity between the targeting and blocking groups of 4 T1 cells was significantly greater than that in HEK293 cells (P < 0.05). The in vitro MRI demonstrated a more remarkable T2 reduction in 4 T1 cells than in HEK293 cells (P < 0.001). The in vivo MRI of 4 T1 xenograft tumor-bearing nude mice showed significant T2 reduction in tumors compared to controls. Prussian blue staining further confirmed that αvβ6 integrin-targeted nanoparticles were specifically accumulated in 4 T1 tumors and notably fewer nanoparticles were detected in 4 T1 tumors of mice injected with control USPIO and HEK293 tumors of mice administered cFK-9-USPIO. Conclusions Integrin αvβ6-targeted nanoparticles have great potential for use in the detection of αvβ6-overexpressed breast cancer with MR molecular imaging.

scholarly journals Monitoring Tritrophic Biocontrol Interactions Between Bacillus spp., Fusarium oxysporum f. sp. cubense, Tropical Race 4, and Banana Plants in vivo Based on Fluorescent Transformation System

2021 ◽  
Vol 12 ◽  
Author(s):  
Ping He ◽  
Shu Li ◽  
Shengtao Xu ◽  
Huacai Fan ◽  
Yongfen Wang ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Laser Scanning Confocal Microscopy ◽  
Transformation System ◽  
Scanning Confocal Microscopy ◽  
Bacillus Spp ◽  
Green Fluorescent ◽  
Race 4

Bacillus spp. is effective biocontrol agents for Fusarium wilt of banana (FWB), tropical race 4 (TR4). This study explores the colonization by Bacillus subtilis, Bacillus velezensis, and Bacillus amyloliquefaciens of host banana plants and elucidates the mechanism of antagonistic TR4 biocontrol. The authors selected one B. subtilis strain, three B. velezensis strains, and three B. amyloliquefaciens strains that are proven to significantly inhibit TR4 in vitro, optimized the genetic transformation conditions and explored their colonization process in banana plants. The results showed that we successfully constructed an optimized fluorescent electro-transformation system (OD600 of bacteria concentration=0.7, plasmid concentration=50ng/μl, plasmid volume=2μl, transformation voltage=1.8kV, and transformation capacitance=400Ω) of TR4-inhibitory Bacillus spp. strains. The red fluorescent protein (RFP)-labeled strains were shown to have high stability with a plasmid-retention frequency above 98%, where bacterial growth rates and TR4 inhibition are unaffected by fluorescent plasmid insertion. In vivo colonizing observation by Laser Scanning Confocal Microscopy (LSCM) and Scanning Electron Microscopy (SEM) showed that Bacillus spp. can colonize the internal cells of banana plantlets roots. Further, fluorescent observation by LSCM showed these RFP-labeled bacteria exhibit chemotaxis (chemotaxis ratio was 1.85±0.04) toward green fluorescent protein (GFP)-labeled TR4 hyphae in banana plants. We conclude that B. subtilis, B. velezensis, and B. amyloliquefaciens can successfully colonize banana plants and interact with TR4. Monitoring its dynamic interaction with TR4 and its biocontrol mechanism is under further study.

scholarly journals Studying the Neurovascular Unit: An Improved Blood–Brain Barrier Model

2009 ◽  
Vol 29 (12) ◽  
pp. 1879-1884 ◽  
Author(s):  
Christoph M Zehendner ◽  
Heiko J Luhmann ◽  
Christoph RW Kuhlmann
Keyword(s):  
Blood Brain Barrier ◽  
Laser Scanning ◽  
Neurovascular Unit ◽  
Cell Types ◽  
Laser Scanning Confocal Microscopy ◽  
Brain Barrier ◽  
Scanning Confocal Microscopy ◽  
Blood Brain

The blood–brain barrier (BBB) closely interacts with the neuronal parenchyma in vivo. To replicate this interdependence in vitro, we established a murine coculture model composed of brain endothelial cell (BEC) monolayers with cortical organotypic slice cultures. The morphology of cell types, expression of tight junctions, formation of reactive oxygen species, caspase-3 activity in BECs, and alterations of electrical resistance under physiologic and pathophysiological conditions were investigated. This new BBB model allows the application of techniques such as laser scanning confocal microscopy, immunohistochemistry, fluorescent live cell imaging, and electrical cell substrate impedance sensing in real time for studying the dynamics of BBB function under defined conditions.

Morphological assessment of the development of multinucleated giant cells in glioma by using mitosis-specific phosphorylated antibodies

2003 ◽  
Vol 98 (4) ◽  
pp. 854-859 ◽  
Author(s):  
Kenkou Maeda ◽  
Masaaki Mizuno ◽  
Toshihiko Wakabayashi ◽  
Syuntarou Takasu ◽  
Tetsurou Nagasaka ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Giant Cells ◽  
Laser Scanning Confocal Microscopy ◽  
Multinucleated Giant Cells ◽  
Content Type ◽  
Scanning Confocal Microscopy ◽  
Drug Treatments ◽  
Fine Print

Object. The nature and origin of multinucleated giant cells in glioma have not been made clear. To investigate the phosphorylation of intermediate filaments, the authors studied multinucleated giant cells in vitro and in vivo by using mitosis-specific phosphorylated antibodies. Methods. Cultured human glioma cells were immunostained with monoclonal antibodies (mAbs) 4A4, KT13, and TM71, which recognized the phosphorylation of vimentin at Ser55, glial fibrillary acidic protein at Ser13, and vimentin at Ser71, respectively. Subsequently, the nature of multinucleated giant cells was investigated using laser scanning confocal microscopy. In addition, paraffin-embedded tissue sections obtained in three patients with giant cell glioblastoma were also investigated. Multinucleated giant cells were immunoreacted with the mAb 4A4 and not with KT13 and TM71 in vitro and in vivo. In addition, the authors obtained these results in multinucleated giant cells under natural conditions, without drug treatments. Conclusions. Findings in this investigation indicated that multinucleated giant cells are those remaining in mitosis between metaphase and telophase, undergoing neither fusion nor degeneration.

scholarly journals Advanced Static and Dynamic Fluorescence Microscopy Techniques to Investigate Drug Delivery Systems

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 861
Author(s):  
Jacopo Cardellini ◽  
Arianna Balestri ◽  
Costanza Montis ◽  
Debora Berti
Keyword(s):  
Drug Delivery ◽  
Fluorescence Microscopy ◽  
Drug Delivery Systems ◽  
Laser Scanning ◽  
Super Resolution ◽  
Laser Scanning Confocal Microscopy ◽  
Delivery Systems ◽  
Scanning Confocal Microscopy ◽  
Biological Phenomena

In the past decade(s), fluorescence microscopy and laser scanning confocal microscopy (LSCM) have been widely employed to investigate biological and biomimetic systems for pharmaceutical applications, to determine the localization of drugs in tissues or entire organisms or the extent of their cellular uptake (in vitro). However, the diffraction limit of light, which limits the resolution to hundreds of nanometers, has for long time restricted the extent and quality of information and insight achievable through these techniques. The advent of super-resolution microscopic techniques, recognized with the 2014 Nobel prize in Chemistry, revolutionized the field thanks to the possibility to achieve nanometric resolution, i.e., the typical scale length of chemical and biological phenomena. Since then, fluorescence microscopy-related techniques have acquired renewed interest for the scientific community, both from the perspective of instrument/techniques development and from the perspective of the advanced scientific applications. In this contribution we will review the application of these techniques to the field of drug delivery, discussing how the latest advancements of static and dynamic methodologies have tremendously expanded the experimental opportunities for the characterization of drug delivery systems and for the understanding of their behaviour in biologically relevant environments.

The Growth Inhibition of Targeted Pluronic/Formononetin Nanocomposite on Oral Squamous Cell Carcinoma In Vitro

2020 ◽  
Vol 12 (8) ◽  
pp. 1022-1029
Author(s):  
Ming Liu ◽  
Chen Lin ◽  
Xiaoqing Huang ◽  
Yuxiang Lin
Keyword(s):  
Squamous Cell Carcinoma ◽  
Oral Squamous Cell Carcinoma ◽  
Growth Inhibition ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Laser Scanning ◽  
Critical Concentration ◽  
Laser Scanning Confocal Microscopy

Natural flavonoid formononetin (FN) has anticancer effects, but the hydrophobic structure, characteristics of the short half-life in vivo, limiting its clinical wide-ranging application. In this study, FN loaded Pluronic (PF)@folic acid (FA) micelles (FN-PF@FA), were prepared to improve the solubility, bioavailability and targeting. FA coupling PF was prepared by carbodiimide crosslinker chemical method, FN-PF@FA micelles were prepared by modified film hydration method, and compared the antitumor activity of FN loaded micelles with free FN In Vitro. The spherical smooth surface of FN-PF@FA micelles had smaller particle size (112.3±5.3 nm), high encapsulation efficiency (86.14±2.68%), high negative zeta potential (-25.8±0.57 mV), low critical concentration CMC (0.03 mg/mL), and better sustained release profile. In addition, FN-PF@FA micelles have a positive targeting effect on oral squamous cell carcinoma cells (SCC3). In 48 hours, the growth inhibition of 50% (GI50) was 28.6±1.2 μg/mL for FN and 17.4±0.78 μg/mL for FN-PF, the dose dropped by nearly 38.46%. In addition, the GI50 value of FN-PF@FA was 9.5±0.3 μg/mL, 66.43% lower than FN and 44.83% lower than FN-PF. Furthermore, the laser scanning confocal microscopy revealed that the conjugation of FA significantly improves the active targeting ability of micelles. FN-PF@FA micelles have the potential to target the release of anticancer drugs with higher bioavailability, further provides a new avenue for the application of traditional Chinese medicine extract in oral malignant tumor.

Synthesis, singlet oxygen generation, photocytotoxicity and subcellular localization of azobisporphyrins as potentially photodynamic therapeutic agents in vitro cell study

2017 ◽  
Vol 21 (02) ◽  
pp. 122-127 ◽  
Author(s):  
Yunman Zheng ◽  
Sizhe Zhu ◽  
Lijun Jiang ◽  
Fengshou Wu ◽  
Chi Huang ◽  
...  
Keyword(s):  
Singlet Oxygen ◽  
Hela Cells ◽  
Cellular Localization ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Confocal Laser Scanning Microscope ◽  
Confocal Laser ◽  
Oxygen Generation ◽  
Scanning Confocal Microscopy

Three azobisporphyrins (Por1, Por2 and Por3) were synthesized by coupling two molecules of (4-nitrophenyl/pyridyl) porphyrins in the presence of KOH/butanol. The structures of porphyrins were confirmed by UV, IR, NMR and mass spectra and elemental analysis. With tetraphenylporphyrin (H2TPP) as a control, the singlet oxygen (1O[Formula: see text] generation of porphyrins was evaluated through 1,3-diphenylisobenzofuran (DPBF) method. The order of ability to generate 1O2 for three azobisporphyrins was Por 1 [Formula: see text]Por 2 > Por 3[Formula: see text] H2TPP. The photocytotoxicity and sub-cellular localization of azobisporphyrins over Hela cells were studied through MTT analysis and confocal laser scanning microscope, respectively. The results indicated Por 1 and Por 2 displayed the low dark-cytotoxicity, while Por 3 induced a concentration-dependent cytotoxicity to Hela cells with the concentration of porphyrins ranging from 1 to 100 [Formula: see text] M. With the light dose at 4 J/cm2, Por 3 killed more than 60% Hela cells at 2 [Formula: see text] M, indicating a high photocytoxicity. As seen from the laser scanning confocal microscopy images, Por 3 was mainly localized in cell membrane, while Por 1 and Por 2 do not displayed significant fluorescent emission in Hela cells. These results suggest the synthesized cationic azobisporphyrin could be used as a potential therapeutic agent for photodynamic therapy of cancers.

2,3′,4,4′,5-Pentachlorobiphenyl Induced Thyrocyte Autophagy by Promoting Calcium Influx via Store-Operated Ca2+ Entry

2020 ◽  
Vol 177 (2) ◽  
pp. 483-493
Author(s):  
Li Wang ◽  
Wenli Xu ◽  
Qi Zhou ◽  
Bojin Xu ◽  
Yunlu Sheng ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Calcium Influx ◽  
Underlying Mechanism ◽  
Laser Scanning Confocal Microscopy ◽  
Thyroid Cell ◽  
Dependent Manner ◽  
Class Iii ◽  
Rat Thyroid

Abstract PCB118, a 2,3′,4,4′,5-pentachlorobiphenyl, has been shown to destroy thyroidal ultrastructure and induce thyrocyte autophagy. Previously, we reported that PCB118 promoted autophagosome formation in vivo and in vitro, but more details remain to be revealed. To explore the underlying mechanism by which PCB118 regulates thyrocyte autophagy, Fischer rat thyroid cell line-5 (FRTL-5) cells were exposed to different doses of PCB118 at 0, 0.25, 2.5, and 25 nM for 0–48 h. Western blot analysis of autophagy-related proteins P62, BECLIN1, and LC3 demonstrated that PCB118 induced autophagy formation in dose- and time-dependent manner. Moreover, laser scanning confocal microscopy and flow cytometry showed PCB118 treatment led to time- and dose-dependent increase in intracellular calcium concentration ([Ca2+]i). Additionally, PCB118 promoted store-operated Ca2+ entry (SOCE) channel followed by significant increase of ORAI1 and STIM1 protein levels. On the other hand, PCB118 induced thyroidal autophagy via class III β-tubulin (TUBB3)/death-associated protein kinase 2 (DAPK2)/myosin regulatory light chain (MRLC)/autophagy-related 9A (ATG9A) pathway in FRTL-5 cells. Pretreatment with SOCE inhibitor SKF96365 reduced cytosolic Ca2+, ORAI1, STIM1, and BECLIN1 levels as well as LC3 II/LC3 I ratio, while increased P62 expression. SKF96365 also inhibited TUBB3/DAPK2/MRLC/ATG9A pathway in FRTL-5 cells treated by PCB118. Our results provide evidence that PCB118 may induce thyroidal autophagy through TUBB3-related signaling pathway, and these effects are likely to be regulated by calcium influx via SOCE channel.

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