Quantitative intracellular magnetic assays on live cells

描述（由申请人提供）：MagneSensors的项目目标是开发定量细胞内磁性测定法，用于快速测定活细胞中的蛋白质水平。该计划将使用超灵敏的磁性传感器来实时检测与靶蛋白特异性结合的磁性纳米颗粒标签。细胞内检测将利用磁检测的两个关键优势。首先，未结合的磁性纳米颗粒标记不给出任何信号，因此不必分离（洗涤）。第二，细胞内没有磁干扰源。虽然一些竞争技术可以进行定量分析或分析活细胞，但两者都很难做到。具体的I期目标是：1）证明使用细胞内磁性测定作为实时工具来定量细胞穿透肽将货物递送到活细胞的细胞质中的有效性的能力2）证明以高灵敏度（信噪比=5的<5，000个细胞）定量活细胞中细胞内蛋白质的表达的能力。细胞穿透肽将用于将与检测抗体缀合的磁性纳米颗粒递送到细胞中。对于基于TAT的细胞穿透肽，货物通过专门形式的内吞作用进入细胞，并且在被释放到细胞质中之前首先进入称为巨胞饮体的囊泡。货物释放的功效随不同的递送肽而变化，并且可能受多种因素影响。第一个目标是显示货物释放到细胞质中的速率可以在活细胞上实时定量。将利用基于磁性纳米颗粒货物的递送和细胞质释放的简单模型系统，并且将随时间测量抗微管蛋白-细胞穿透肽-磁性纳米颗粒与微管结构的结合。主要目标是确定磁性测定可以作为一种有用的工具来确定货物递送到细胞中的有效性。第二个目的是证明可以高灵敏度定量测量细胞内蛋白质（例如，肝细胞中的P450同工酶，其对药物开发有用）的表达。体外细胞内测定将使用改进的细胞穿透肽如TAT-HA 2。一个关键的目标是显示由于细胞内和细胞表面上的非特异性结合的纳米颗粒标记的背景是最小的，从而能够检测少量（<5，000）活细胞。注意，高灵敏度将允许测量以低浓度表达的蛋白质。长期目标是向市场推出一种新的磁性仪器和试剂平台，可以使药物输送和药物开发中的治疗应用受益。定量测量活细胞内蛋白质水平的能力可以使广泛的应用受益，特别是在治疗学中。对于药物递送，可以准确地确定细胞内运输的治疗剂的释放速率的工具将是特别有价值的，因为递送在药物的有效性中起主要作用。对于药物开发，分析关键蛋白质水平对药物的反应的能力可以帮助生产更安全的药物，并减少不良反应，这是美国死亡的主要原因之一。

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.