Role of surface charge in the internalization mechanisms of carboxy-methyl dextra

描述（由申请人提供）：基本了解纳米颗粒的物理化学性质（如大小、电荷、亲水性等）在细胞内化和细胞内定位中的作用，是合理设计和优化纳米颗粒用于成像、传感、药物或DNA递送、细胞分选和许多其他临床应用的必要条件。这项应用的长期目标是阐明纳米粒子的物理化学性质与细胞内纳米粒子的内化和命运之间的关系。作为第一个具体目标，我们将改进方法，使用已建立的药理学药物系统地抑制体外细胞的分子内化途径，并证明这些方法可用于阐明羧甲基葡聚糖包被的磁铁矿纳米颗粒内化到上皮来源的caco2（人类结肠癌来源）和MFC-7（人类乳腺癌来源）癌细胞系的机制。待改进的方法将允许区分主动和被动运输机制、网格蛋白依赖途径（以及受体介导的内吞作用和液相内吞作用的细分）和网格蛋白独立途径（以及腔泡和脂质筏依赖的内吞作用的细分）。作为第二个具体目标，表面电荷对羧甲基葡聚糖包被纳米颗粒的内在化和细胞内定位的作用将通过使用类似大小的磁铁矿纳米颗粒来评估，这些纳米颗粒包被相同分子量的羧甲基葡聚糖，但羧酸取代程度不同。通过上述每个分子途径的纳米颗粒内化将使用共聚焦激光扫描显微镜进行定性评估，并使用电感耦合等离子体质谱进行定量评估。通过本文所提出的工作所改进的方法应该适用于广泛的无机核/有机壳纳米颗粒和广泛的靶细胞的纳米颗粒内化机制的系统研究。获得表面电荷在羧甲基右旋糖酐包被纳米颗粒内化过程中的机制和作用的具体信息，将适用于合理设计磁性纳米颗粒，可能提高其作为MRI造影剂、磁性靶向药物递送载体和磁流体热疗治疗癌症的功效。

DESCRIPTION (provided by applicant): Fundamental understanding of the role of nanoparticle physicochemical properties (such as size, charge, hydrophilicity, etc.) on internalization into cells and intracellular localization is a requirement for the rational design and optimization of nanoparticles for imaging, sensing, drug or DNA delivery, cell sorting, and many other clinical applications. The long term objective of this application is to elucidate the relationship between nanoparticle physicochemical properties and internalization and fate of nanoparticles in cells. As a first specific aim, methods will be refined to use established pharmacological agents to systematically inhibit molecular internalization pathways of cells in vitro and demonstrate that such methods can be applied to elucidate the mechanism of internalization of carboxy-methyl dextran coated magnetite nanoparticles into the cancer cell lines of epithelial origin Caco-2 (human colon cancer derived) and MFC-7 (human breast cancer derived). The methods to be refined will permit discrimination between active and passive transport mechanisms, clathrin- dependent pathways (and the sub-divisions of receptor mediated endocytosis and fluid phase endocytosis), and clathrin-independent pathways (and the subdivisions of caveolar and lipid raft-dependent endocytosis). As a second specific aim, the role of surface charge on internalization and intracellular localization of carboxy- methyl dextran coated nanoparticles will be assessed by using magnetite nanoparticles of similar size, coated with carboxy-methyl dextran of the same molecular weight but varying degrees of carboxylic acid substitution. Nanoparticle internalization through each of the above-mentioned molecular pathways will be assessed qualitatively using confocal laser scanning microscopy and quantitatively using inductively coupled plasma mass spectrometry. The methods to be refined through the proposed work should find applicability in systematic study of nanoparticle internalization mechanisms for a wide array of inorganic core/organic shell nanoparticles and a wide array of target cells. The specific information to be obtained on mechanisms and effect of surface charge in internalization of carboxy-methyl dextran coated nanoparticles will be applicable to the rational design of magnetic nanoparticles with potentially improved efficacy as MRI contrast agents, magnetically targeted drug delivery vectors, and in magnetic fluid hyperthermia treatment of cancer. PUBLIC HEALTH RELEVANCE: The relationship between nanoparticle physicochemical properties and internalization in cells is a key step in developing medical applications of magnetic nanoparticles, such as MRI contrast agents, sensing, drug delivery, and magnetic fluid hyperthermia cancer treatment. The proposed work will systematically study internalization mechanisms of magnetic nanoparticles in cells and determine the effect of surface charge on rate of internalization and intracellular localization of nanoparticles. The methods refined in this work will also impact study and development of other types of polymer coated inorganic core nanoparticles for medical applications.