Quantitative intracellular magnetic assays on live cells

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DESCRIPTION (provided by applicant): MagneSensors' program goal is to develop quantitative intracellular magnetic assays for rapid determination of protein levels in live cells. The program will use ultra-sensitive magnetic sensors to perform real-time detection of magnetic nanoparticle labels that are specifically bound to target proteins. The intracellular assays will leverage two key advantages of magnetic detection. First, unbound magnetic nanoparticle labels do not give any signal and hence do not have to be separated (washed). Second, there are no magnetic interference sources within cells. While a few competing technologies can perform quantitative analysis or analyze live cells, it is very difficult to do both. The specific Phase I objectives are: 1) demonstrate capability of using intracellular magnetic assays as a real-time tool to quantify the effectiveness of cell-penetrating peptides to deliver cargo into the cytoplasm of live cells 2) demonstrate ability to quantify expression of an intracellular protein in live cells at high sensitivity (<5,000 cells with signal-to-noise =5). A cell-penetrating peptide will be used to deliver the magnetic nanoparticles conjugated with a detect antibody into the cell. For TAT-based cell-penetrating peptides, the cargo enters the cell via a specialized form of endocytosis and first goes into vesicles called macropinosomes before being released into the cytoplasm. The efficacy of cargo release varies with different delivery peptides and may be influenced by a variety of factors. The first objective is to show that the rate of cargo release into the cytoplasm can be quantified on live cells in real-time. A simple model system based on the delivery and cytoplasmic release of magnetic nanoparticle cargo will be utilized and the binding of anti tubulin-cell penetrating peptide-magnetic nanoparticles to microtubule structures will be measured over time. The main goal is to establish that magnetic assays can serve as a useful tool to determine the effectiveness of cargo delivery into cells. The second objective is to demonstrate that the expression of an intracellular protein (e.g. P450 isoenzyme in liver cells, which is useful for drug development) can be quantitatively measured with high sensitivity. The in vitro intracellular assay will use an improved cell-penetrating peptide such as TAT-HA2. A key goal is to show that the background due to non-specifically bound nanoparticle labels inside the cell and on the cell surface is minimal, enabling detection on a small number (<5,000) of live cells. Note that high sensitivity would permit measurement of proteins expressed in low concentrations. The longer-term objective is to bring to market a new magnetic instrumentation and reagent platform that can benefit therapeutic applications in drug delivery and drug development. The capability to quantitatively measure protein levels inside live cells can benefit a wide range of applications, particularly in therapeutics. For drug delivery, a tool that can accurately determine the rate of release of a therapeutic transported inside a cell would be especially valuable, as the delivery plays a major role in the effectiveness of a drug. For drug development, the ability to analyze the levels of key proteins in response to a drug could allow help produce safer drugs and reduce the adverse reactions that are one of the leading causes of death in the U.S.