Diaminobenzidine photoconversion is a suitable tool for tracking the intracellular location of fluorescently labelled nanoparticles at transmission electron microscopy

Malatesta, Manuela; Giagnacovo, Marzia; Costanzo, Manuela; Conti, Bice; Genta, Ida; Dorati, Rossella; Galimberti, V.; Biggiogera, Marco; Zancanaro, Carlo

doi:10.4081/ejh.2012.20

Cited by 45 publications

(58 citation statements)

References 40 publications

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“…Now, we aimed at depicting the molecular events leading to HMA cytotoxicity. First, we exploited the fluorescent properties of HMA [7] using the diaminobenzidine (DAB) photo-oxidation technique, which is based on the formation of an electron-dense osmiophilic product that precipitates in close proximity to the fluorophore, thereby allowing its ultrastructural detection [15, 19]. Electron microscopy analysis of HMA-treated HCT-116 colon carcinoma cells revealed electron-dense black spots within the cytoplasm, enclosed within vesicles (red*), which are present only as an effect of HMA, given that in untreated cells no evidence of roundish deposit of electron-dense products was found (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Molecular features of the cytotoxicity of an NHE inhibitor: Evidence of mitochondrial alterations, ROS overproduction and DNA damage

et al. 2016

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BackgroundNH exchangers (NHEs) play a crucial role in regulating intra/extracellular pH, which is altered in cancer cells, and are therefore suitable targets to alter cancer cell metabolism in order to inhibit cell survival and proliferation. Among NHE inhibitors, amiloride family members are commonly used in clinical practice as diuretics; we focused on the amiloride HMA, reporting a net cytotoxic effect on a panel of human cancer cell lines; now we aim to provide new insights into the molecular events leading to cell death by HMA.MethodsColon cancer cell lines were treated with HMA and analysed with: morphological and cellular assays for cell viability and death, and autophagy; biochemical approaches to evaluate mitochondrial function and ROS production; in situ detection of DNA damage; molecular tools to silence crucial autophagy/necroptosis factors.ResultsHMA affects cellular morphology, alters mitochondrial structure and function, causes an increase in ROS, which is detrimental to DNA integrity, stimulates poly(ADP-ribose) synthesis, activates RIPK3-dependent death and triggers autophagy, which is unable to rescue cell survival. These features are hot points of an intricate network of processes, including necroptosis and autophagy, regulating the homeostasis between survival and death.ConclusionOur results allow the identification of multiple events leading to cell death in cancer cells treated with HMA. The here-defined intricate network activated by HMA could be instrumental to selectively target the key players of each pathway in the attempt to improve the global response to HMA. Our data could be the starting point for developing a newly designed targeted therapy.

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Section: Resultsmentioning

confidence: 99%

“…The samples were finally examined and photographed with a Zeiss EM 900 electron microscope at 80 KV (Carl Zeiss, Jena, Germany). The micrographs were then developed and digitalised [15]. …”

Section: Methodsmentioning

confidence: 99%

Molecular features of the cytotoxicity of an NHE inhibitor: Evidence of mitochondrial alterations, ROS overproduction and DNA damage

et al. 2016

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“…TEM images showed that labeled ASCs incorporate USPIO by endocytic mechanisms, as already reported. 29 It has been demonstrated that nanoparticles internalized by endocytosis often accumulate inside multivesicular bodies, 30 which, in turn, may fuse with the plasma membrane thus releasing their cargo (exosomes). 31 It is likely that USPIO undergo the same secretion process of the endosomal system.…”

Section: Discussionmentioning

confidence: 99%

Magnetic resonance imaging of ultrasmall superparamagnetic iron oxide-labeled exosomes from stem cells: a new method to obtain labeled exosomes

Busato

Bonafede

Bontempi

et al. 2016

IJN

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Purpose Recent findings indicate that the beneficial effects of adipose stem cells (ASCs), reported in several neurodegenerative experimental models, could be due to their paracrine activity mediated by the release of exosomes. The aim of this study was the development and validation of an innovative exosome-labeling protocol that allows to visualize them with magnetic resonance imaging (MRI). Materials and methods At first, ASCs were labeled using ultrasmall superparamagnetic iron oxide nanoparticles (USPIO, 4–6 nm), and optimal parameters to label ASCs in terms of cell viability, labeling efficiency, iron content, and magnetic resonance (MR) image contrast were investigated. Exosomes were then isolated from labeled ASCs using a standard isolation protocol. The efficiency of exosome labeling was assessed by acquiring MR images in vitro and in vivo as well as by determining their iron content. Transmission electron microscopy images and histological analysis were performed to validate the results obtained. Results By using optimized experimental parameters for ASC labeling (200 µg Fe/mL of USPIO and 72 hours of incubation), it was possible to label 100% of the cells, while their viability remained comparable to unlabeled cells; the detection limit of MR images was of 10 2 and 2.5×10 3 ASCs in vitro and in vivo, respectively. Exosomes isolated from previously labeled ASCs retain nanoparticles, as demonstrated by transmission electron microscopy images. The detection limit by MRI was 3 µg and 5 µg of exosomes in vitro and in vivo, respectively. Conclusion We report a new approach for labeling of exosomes by USPIO that allows detection by MRI while preserving their morphology and physiological characteristics.

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“…However, their low water solubility often leads to decreased transfection efficiency [9,10]. To overcome this problem, chitosan nanoparticles have been linked to different molecules to increase their solubility.…”

Section: Drug and Genetic Materials Delivery To The Cnsmentioning

confidence: 99%

Nanoparticles for Brain-Specific Drug and Genetic Material delivery, Imaging and Diagnosis

Posadas

Monteagudo

Ceña

2016

Nanomedicine (Lond.)

108

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The poor access of therapeutic drugs and genetic material into the central nervous system due to the presence of the blood-brain barrier often limits the development of effective noninvasive treatments and diagnoses of neurological disorders. Moreover, the delivery of genetic material into neuronal cells remains a challenge because of the intrinsic difficulty in transfecting this cell type. Nanotechnology has arisen as a promising tool to provide solutions for this problem. This review will cover the different approaches that have been developed to deliver drugs and genetic material efficiently to the central nervous system as well as the main nanomaterials used to image the central nervous system and diagnose its disorders.

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Diaminobenzidine photoconversion is a suitable tool for tracking the intracellular location of fluorescently labelled nanoparticles at transmission electron microscopy

Cited by 45 publications

References 40 publications

Molecular features of the cytotoxicity of an NHE inhibitor: Evidence of mitochondrial alterations, ROS overproduction and DNA damage

Molecular features of the cytotoxicity of an NHE inhibitor: Evidence of mitochondrial alterations, ROS overproduction and DNA damage

Magnetic resonance imaging of ultrasmall superparamagnetic iron oxide-labeled exosomes from stem cells: a new method to obtain labeled exosomes

Nanoparticles for Brain-Specific Drug and Genetic Material delivery, Imaging and Diagnosis

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