Quantitative intracellular magnetic assays on live cells

活细胞的定量细胞内磁性测定

• 批准号: 7219573
• 负责人: Mark S. DiIorio
• 金额: $ 16.83万
• 依托单位: MAGNESENSORS, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7219573
• 关键词: Address; Adverse reactions; Antibodies; Area; Back; Binding; Biological Assay; Biological Models; Biotechnology; Bypass; Caliber; Cause of Death; Cell Surface Receptors; Cell membrane; Cell surface; Cells; Confocal Microscopy; Cytochrome P450; Cytolysis; Cytoplasm; Data; Detection; Development; Devices; Drug Delivery Systems; Effectiveness; Endocytosis; Enzymes; Figs - dietary; Foundations; Goals; Head; Hepatocyte; High temperature of physical object; Hour; Immunoassay; In Vitro; Institutes; Interleukin-6; Isoenzymes; Kinetics; Knowledge; Label; Lead; Legal patent; Life; Magnetic Resonance Imaging; Magnetism; Marketing; Measurement; Measures; Medical; Melanocytic nevus; Methods; Microtubules; Minor; Mole the mammal; Monoclonal Antibodies; Nature; Noise; Numbers; Pathway interactions; Peptides; Pharmaceutical Preparations; Phase; Play; Principal Investigator; Protein Dynamics; Proteins; Publications; Publishing; Range; Rate; Reaction; Reagent; Relative (related person); Research; Research Personnel; Role; Sampling; Second Messenger Systems; Sensitivity and Specificity; Signal Transduction; Solutions; Son of Sevenless Proteins; Source; Structure; Techniques; Technology; Therapeutic; Time; Tubulin; United States National Aeronautics and Space Administration; United States National Institutes of Health; Vesicle; anticancer research; assay development; base; cell fixing; cellular transduction; commercialization; cost; design; drug development; drug metabolism; experience; improved; in vivo; innovation; instrument; instrumentation; interest; medical schools; nanoparticle; novel; professor; programs; protein expression; receptor; research and development; response; second messenger; sensor; size; superconducting quantum interference device; technology development; tool

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.

描述（由申请人提供）：Magnesensors的程序目标是开发定量的细胞内磁化测定，以快速确定活细胞中的蛋白质水平。该程序将使用超敏感的磁传感器对特定与靶蛋白结合的磁性纳米颗粒标签进行实时检测。细胞内测定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签没有任何信号，因此不必分开（洗涤）。其次，细胞内没有磁干扰源。尽管一些竞争技术可以进行定量分析或分析活细胞，但两者都很难进行。特定的I期目标是：1）证明了使用细胞内磁性测定作为实时工具的能力，以量化细胞穿透肽的有效性，以将货物输送到活细胞的细胞质中2）证明能够量化活细胞中细胞内细胞内细胞内蛋白质在高灵敏度上的表达的能力（<5,000个具有信号至noise信号至noise = 5）的能力。细胞穿透肽将用于输送与检测抗体结合到细胞中的磁性纳米颗粒。对于基于TAT的细胞渗透肽，该货物通过一种专门的内吞作用形式进入细胞，并首先进入称为大型粒细胞的囊泡，然后释放到细胞质中。货物释放的功效随着递送肽的不同而有所不同，可能受到多种因素的影响。第一个目标是表明可以实时在活细胞上量化货物释放到细胞质中的速度。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，并将随着时间的推移测量抗微管蛋白细胞穿透性肽纳米粒子与微管结构的结合。主要目标是确定磁性测定可以作为确定货物递送到细胞的有效性的有用工具。第二个目标是证明细胞内蛋白的表达（例如肝细胞中的p450同工酶，对药物发育有用），可以用高灵敏度进行定量测量。体外细胞内测定将使用改进的细胞穿透肽，例如Tat-Ha2。一个关键目标是表明，由于细胞内部和细胞表面的非特异性结合纳米颗粒标签引起的背景是最小的，因此可以在少量（<5,000）的活细胞上检测。请注意，高灵敏度将允许以低浓度表达的蛋白质测量。长期目的是推销一种新的磁性仪器和试剂平台，该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，尤其是在治疗剂中。对于药物输送，可以准确确定在细胞内运输的治疗性释放速率的工具特别有价值，因为递送在药物的有效性中起着重要作用。对于药物开发，分析响应药物的关键蛋白水平的能力可以帮助产生更安全的药物并减少不良反应，而不良反应是美国主要的死亡原因之一