Methods for Isolation and Identification of Nanoparticle-Containing Subcellular Compartments
Nanoparticle-based drug delivery systems have considerable potential for improvement of drug stability, bioavailability, and reduced dosing frequency. Important technological advantages of nanoparticles include high carrier capacity across biological memb
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Introduction A wide variety of nanoparticles have been developed for the cellular delivery of various therapeutic compounds and the potential clinical benefits of these particles are great (1, 2). However, very little is known about the subcellular distribution of nanoparticles in the targeted cells. This information is necessary if we are to explain how nanoparticles function on a subcellular level and to identify any potential sources of cellular toxicity. In order to accomplish this, a method must be used that can simultaneously allow for the isolation and subsequent identification of proteins that interact with a nanoparticle while it is in a cell. Here, we demonstrate that the proteins that come into contact with a nanoparticle can be individually labeled, isolated, and then identified by liquid chromatography–mass spectrometry (LC-MS/MS). This relatively simple method involves four basic steps: (1) labeling of the nanoparticles
Equal contributions were made by Ari Nowacek and Irena Kadiu. Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology, vol. 991, DOI 10.1007/978-1-62703-336-7_6, © Springer Science+Business Media New York 2013
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with a visible dye, (2) treatment of cells with the nanoparticles, (3) isolation of nanoparticle-laden subcellular compartments on a sucrose gradient, and (4) identification of the proteomes of subcellular compartments by LC/MS-MS. This method provides the user with a broad view of the subcellular distribution of nanoparticles within the same experiment. It is appropriate for use by researchers who do not know the fate of their nanoformulations within the targeted cells or their mechanisms of release. It can also be used successfully to identify the subcellular trafficking pathways of crystalline antiretroviral nanoparticles in human monocyte-derived macrophages (3). Alternative approaches such as immunostaining and confocal imaging of every cellular organelle and internalized nanoparticles as well as measurement of their fluorescence overlap are time consuming and costly.
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Materials Prepare all solutions using ultrapure water (prepared by purifying deionized water to attain a sensitivity of 18 MΩ cm at 25°C) and analytical grade reagents. Prepare and store all reagents at room temperature (unless indicated otherwise). Diligently follow all waste disposal regulations when disposing waste materials.
2.1 Components to Label Nanoparticles
1. Crystalline nanoparticles (see Note 1). 2. Coomassie Brilliant Blue R250 (CBB) (see Note 2). 3. Sterile 1× phosphate buffered saline (PBS). 4. 0.5 or 1.7 mL microcentrifuge tubes. 5. Microcentrifuge tube tumbler rotator. 6. Table-top refrigerated centrifuge that can reach 20,000 × g. 7. Sonicator with probe. 8. Method to measure nanoparticle size and charge (see Note 3).
2.2 Cellular Treatment Components
1. Cells in vitro (see Note 4). 2. Cell incubator. 3. Serum-free DMEM (or other appropriate serum-free cell culture medium). 4. Labeled
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