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Temperature-independent porous nanocontainers for single-molecule fluorescence studies.

ABSTRACT: In this work, we demonstrate the capability of using lipid vesicles biofunctionalized with protein channels to perform single-molecule fluorescence measurements over a biologically relevant temperature range. Lipid vesicles can serve as an ideal nanocontainer for single-molecule fluorescence measurements of biomacromolecules. One serious limitation of the vesicle encapsulation method has been that the lipid membrane is practically impermeable to most ions and small molecules, limiting its application to observing reactions in equilibrium with the initial buffer condition. To permeabilize the barrier, Staphylococcus aureus toxin ?-hemolysin (aHL) channels have been incorporated into the membrane. These aHL channels have been characterized using single-molecule fluorescence resonance energy transfer signals from vesicle-encapsulated guanine-rich DNA that folds in a G-quadruplex motif as well as from the Rep helicase-DNA system. We show that these aHL channels are permeable to monovalent ions and small molecules, such as ATP, over the biologically relevant temperature range (17-37 °C). Ions can efficiently pass through preformed aHL channels to initiate DNA folding without any detectable delay. With addition of the cholesterol to the membrane, we also report a 35-fold improvement in the aHL channel formation efficiency, making this approach more practical for wider applications. Finally, the temperature-dependent single-molecule enzymatic study inside these nanocontainers is demonstrated by measuring the Rep helicase repetitive shuttling dynamics along a single-stranded DNA at various temperatures. The permeability of the biofriendly nanocontainer over a wide range of temperature would be effectively applied to other surface-based high-throughput measurements and sensors beyond the single-molecule fluorescence measurements.

SUBMITTER: Ishitsuka Y

PROVIDER: S-EPMC3058344 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Temperature-independent porous nanocontainers for single-molecule fluorescence studies.

Ishitsuka Yuji Y Okumus Burak B Arslan Sinan S Chen Kok Hao KH Ha Taekjip T

Analytical chemistry 20101101 23

In this work, we demonstrate the capability of using lipid vesicles biofunctionalized with protein channels to perform single-molecule fluorescence measurements over a biologically relevant temperature range. Lipid vesicles can serve as an ideal nanocontainer for single-molecule fluorescence measurements of biomacromolecules. One serious limitation of the vesicle encapsulation method has been that the lipid membrane is practically impermeable to most ions and small molecules, limiting its applic ...[more]

PMID: 21038883

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Project description:Nanomaterials are a central pillar in modern medicine. They are thought to optimize drug delivery, enhance therapeutic efficacy, and reduce side-effects. To foster this technology, analytical methods are needed to validate not only the localization and distribution of these nanomaterials, but also their compatibility with cells, drugs, and drug release. In the present work, we assessed nanoparticles based on porous silicon (pSiNPs) loaded with the clinically used tyrosine kinase inhibitor sunitinib for their effectiveness of drug delivery, release, and toxicity in colon cancer cells (HCT 116 cells) and cardiac myoblast cells (H9c2) using Raman micro-spectroscopy, high-resolution fluorescence microscopy, along with biological methods for toxicological effects. We produced pSiNPs with a size of about 100 nm by grinding mesoporous silicon layers. pSiNPs allowed an effective loading of sunitinib due to their high porosity. Photoluminescence properties of the nanoparticles within the visible spectrum allowed the visualization of their uptake in cardiac cells. Raman micro-spectroscopy allowed not only the detection of the uptake and distribution of pSiNPs within the cells via a characteristic silicon Raman band at about 518-520 cm-1, but also the localization of the drug based on its characteristic molecular fingerprints. Cytotoxicity studies by Western blot analyses of apoptotic marker proteins such as caspase-3, and the detection of apoptosis by subG1-positive cell fractions in HCT 116 and MTT analyses in H9c2 cells, suggest a sustained release of sunitinib from pSiNPs and delayed cytotoxicity of sunitinib in HCT 116 cells. The analyses in cardiac cells revealed that pSiNPs are well tolerated and that they may even protect from toxic effects in these cells to some extent. Analyses of the integrity of mitochondrial networks as an early indicator for apoptotic cellular effects seem to validate these observations. Our study suggests pSiNPs-based nanocontainers for efficient and safe drug delivery and Raman micro-spectroscopy as a reliable method for their detection and monitoring. Thus, the herein presented nanocontainers and analytical methods have the potential to allow an efficient advancement of nanoparticles for targeted and sustained intracellular drug release that is of need, e.g., in chronic diseases and for the prevention of cardiac toxicity.

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Temperature-independent porous nanocontainers for single-molecule fluorescence studies.

Publications

Temperature-independent porous nanocontainers for single-molecule fluorescence studies.

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