Magnetic nanobeads for cancer cell selection

描述（由申请人提供）：本提案的目的是开发用于四重磁分选（QMS）的高分辨率磁性纳米颗粒。QMS是一种流通式免疫磁性分离系统，可以提供血液以及其他生物流体中循环癌细胞的灵敏富集。通过QMS的最佳细胞分离需要具有高磁化率、窄粒度分布和高密度抗体附着位点的免疫磁性颗粒。目前商业化的免疫磁珠要么太大，缺乏尺寸均匀性，要么具有太低的磁化率。我们将利用纳米颗粒技术的最新进展对磁性免疫珠进行必要的改进，并通过从复杂的培养细胞群体中富集卵巢癌细胞来评估其性能。在具体目标1中，我们将使用溶胶-凝胶方法合成并物理表征单分散顺磁性纳米颗粒以封装Fe纳米晶体。这些纳米颗粒将具有均匀的尺寸分布和形状，并且具有高重量百分比的铁（里程碑1）。在具体目标2中，我们将使用硅氧烷处理和异双功能偶联剂来为抗体和链霉亲和素创建高密度连接位点（里程碑2）。将分析所得颗粒的颗粒场相互作用参数值，这是磁性纳米颗粒在细胞分离中有效性的主要决定因素（里程碑3）。最后，在具体目标3中，我们将评估我们的磁性纳米颗粒用于免疫磁性检测和QMS分离卵巢肿瘤细胞。将人卵巢四癌PA-1肿瘤细胞稀释至全血中，并使用针对TAG-72（卵巢癌细胞上存在的表面表达蛋白）的抗体进行QMS。证明全血细胞中肿瘤细胞的检测将作为本项目的原理证明。

DESCRIPTION (provided by applicant): The aim of this proposal is to develop high-resolution magnetic nanoparticles for use in Quadruple Magnetic Sorting (QMS). QMS is a flow-through immunomagnetic separation system that can provide sensitive enrichment of circulating cancer cells in blood as well as other biological fluids. Optimal cellular separation by QMS requires immunomagnetic particles having high magnetic susceptibility, narrow particle size distribution and high density attachment sites for antibodies. Current commercial immunomagnetic beads are either too large, lack size uniformity, or have too low magnetic susceptibility. We will make the necessary improvements in the magnetic immunobeads using recent advances in nano-particle technology and evaluate their performance by enriching ovarian cancer cells from a complex population of cultured cells. In Specific Aim 1 we will synthesize and physically characterize monodisperse paramagnetic nanoparticles using sol-gel methods to encapsulate Fe nano-crystals. These nanoparticles will have uniform size distribution and shape, and possess a high weight percentage of iron (Milestone 1). In Specific Aim 2 we will use siloxane treatments and heterobifunctional coupling agents to create high-density attachment sites for antibodies and streptavidin (Milestone 2). The resulting particles will be analyzed for their particle field interaction parameter values which is a major determinant of the effectiveness of magnetic nanoparticles in cellular separation (Milestone 3). Finally in Specific Aim 3 we will evaluate our magnetic nanoparticles for immunomagnetic detection and separation of ovarian tumor cells by QMS. The human ovarian tetracarcinoma PA-1 tumor cells will be diluted into whole blood and subjected to QMS using antibodies against TAG-72, a surface-expressed protein present on ovarian cancer cells. Demonstrating the detection of tumor cells in whole blood cells will be taken as the proof of principal for this project.