Dataset Information

Magnetic Separation of Autophagosomes from Mammalian Cells Using Magnetic-Plasmonic Hybrid Nanobeads.

ABSTRACT: Developments in subcellular fractionation strategies have provided the means to analyze the protein and lipid composition of organelles by proteomics. Here, we developed ultrasmall magnetic-plasmonic hybrid nanobeads and applied them to the isolation of autophagosomes by applying a magnetic field. The beads were chemically synthesized and comprised an Ag/FeCo/Ag core/shell/shell structure with a mean diameter of 15 nm. The Ag core and the FeCo shell conferred imaging and magnetic separation capabilities, respectively. The nanobeads were transfected into mammalian cells by lipofection. Thirty minutes after lipofection, the nanobeads colocalized with Vps26 and subsequently with LC3. Cell lysates were prepared at the appropriate time points and were subjected to magnetic separation. The separated fraction contained LC3-II, transferrin receptor, and LAMP2, but not LC3-I, suggesting that autophagosomes engulfing endosomal origin had been isolated. The magnetic separation process was completed in less than 30 min, providing a rapid method for isolation of autophagosomes. The present organelle isolation technique using the hybrid nanobeads with imaging and magnetic separation capabilities is highly promising for isolation of other types of organelles such as endosomes and endosome-related organelles.

SUBMITTER: Takahashi M

PROVIDER: S-EPMC6044991 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Publications

Magnetic Separation of Autophagosomes from Mammalian Cells Using Magnetic-Plasmonic Hybrid Nanobeads.

Takahashi Mari M Mohan Priyank P Mukai Kojiro K Takeda Yuichi Y Matsumoto Takeo T Matsumura Kazuaki K Takakura Masahiro M Arai Hiroyuki H Taguchi Tomohiko T Maenosono Shinya S

ACS omega 20170824 8

Developments in subcellular fractionation strategies have provided the means to analyze the protein and lipid composition of organelles by proteomics. Here, we developed ultrasmall magnetic-plasmonic hybrid nanobeads and applied them to the isolation of autophagosomes by applying a magnetic field. The beads were chemically synthesized and comprised an Ag/FeCo/Ag core/shell/shell structure with a mean diameter of 15 nm. The Ag core and the FeCo shell conferred imaging and magnetic separation capa ...[more]

PMID: 30023731

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Project description:The epidermal growth factor receptor (EGFR) is overexpressed in 80% of non-small cell lung cancer (NSCLC) and is associated with poor survival. In recent years, EGFR-targeted inhibitors have been tested in the clinic for NSCLC. Despite the emergence of novel therapeutics and their application in cancer therapy, the overall survival rate of lung cancer patients remains 15%. To develop more effective therapies for lung cancer we have combined the anti-EGFR antibody (Clone 225) as a molecular therapeutic with hybrid plasmonic magnetic nanoparticles (NP) and tested on non-small cell lung cancer (NSCLC) cells.Cell viability was determined by trypan-blue assay. Cellular protein expression was determined by Western blotting. C225-NPs were detected by electron microscopy and confocal microscopy, and EGFR expression using immunocytochemistry. C225-NP exhibited a strong and selective antitumor effect on EGFR-expressing NSCLC cells by inhibiting EGFR-mediated signal transduction and induced autophagy and apoptosis in tumor cells. Optical images showed specificity of interactions between C225-NP and EGFR-expressing NSCLC cells. No binding of C225-NP was observed for EGFR-null NSCLC cells. C225-NP exhibited higher efficiency in induction of cell killing in comparison with the same amount of free C225 antibody in tumor cells with different levels of EGFR expression. Furthermore, in contrast to C225-NP, free C225 antibody did not induce autophagy in cells. However, the therapeutic efficacy of C225-NP gradually approached the level of free antibodies as the amount of C225 antibody conjugated per nanoparticle was decreased. Finally, attaching C225 to NP was important for producing the enhanced tumor cell killing as addition of mixture of free C225 and NP did not demonstrate the same degree of cell killing activity.We demonstrated for the first time the molecular mechanism of C225-NP induced cytotoxic effects in lung cancer cells that are not characteristic for free molecular therapeutics thus increasing efficacy of therapy against NSCLC.

Dataset Information

Magnetic Separation of Autophagosomes from Mammalian Cells Using Magnetic-Plasmonic Hybrid Nanobeads.

Publications

Magnetic Separation of Autophagosomes from Mammalian Cells Using Magnetic-Plasmonic Hybrid Nanobeads.

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