MRI Contrast Agents for In vivo Monitoring of Stem Cell Differentiation

描述（由申请人提供）：干细胞用于治疗的关键是一种非侵入性检测细胞迁移和分化的方法。基于核磁共振成像的细胞跟踪技术已被用于检测细胞在体内的迁移，但目前还无法检测细胞的分化。这是因为目前用于细胞跟踪的磁性颗粒无论在干细胞内还是在成熟细胞内都会产生相同的MRI对比。在这里，我们建议设计、合成和表征新型生物聚合物封装的金属纳米颗粒，这将使MRI能够在体内无创地检测干细胞分化。这些新粒子的基本原理是这些粒子在酶触发的生物聚合物涂层修饰的基础上实现摩尔弛度的大幅增强的能力。简而言之，即使磁性材料的数量保持不变，磁性颗粒的MRI特性也会因磁芯上的涂层厚度及其聚集状态而发生很大变化。此外，MRI造影剂的水溶性有很大的不同。我们将合成不同种类的生物聚合物包被金属芯粒子，其包被将能够被酶切和去除。生物聚合物涂层将具有生物相容性，同时高度抵抗细胞中的被动降解。此外，对于某些颗粒，金属芯将能够溶解。对这些现象的动态操作将导致弛豫度的大幅增强，从而导致MRI信号的增强。计算机模拟预测弛豫随涂层厚度的变化将指导纳米粒子的制备。然后，我们将测试酶介导的生物聚合物涂层去除是否确实会导致MRI特性的调节。模拟预测，以氧化铁为基础的颗粒r2增加10倍，r2*增加4倍，以锰为基础的颗粒r1增加50倍。这些新型颗粒将成为通过MRI对完整生物体中干细胞分化进行无创可视化的技术核心，并可进一步推广到基因表达监测。在内化进入细胞后，纳米和微粒被隔离在核内体和溶酶体中。虽然已知右旋糖酐包覆的磁性颗粒在这些结构中会缓慢降解，但尚未有研究有意利用这一现象。这项工作的创新之处在于，我们利用这些亚细胞结构内的化学环境作为酶促反应的介质，调节水对金属核的可及性，在某些情况下，溶解它们。这些颗粒的未来实现将利用报告酶与转基因颗粒发生反应，

DESCRIPTION (provided by applicant): Key to the promise of stem cells for therapy is a method for non-invasively detecting cellular migration and differentiation. MRI-based cell tracking using magnetic particles has been utilized to detect cell migration in vivo, however, it i currently incapable of detecting cell differentiation. This is because the magnetic particles currently used for cell tracking create the same MRI contrast whether they are inside a stem cell or mature cell. Here we propose to design, synthesize and characterize novel classes of biopolymer encapsulated metallic nanoparticles which will enable the use of MRI to non-invasively detect stem cell differentiation, in vivo. The underlying principal of these new particls is the ability of these particles to achieve large enhancements in molar relaxivity based upon enzyme-triggered modification of the biopolymer coat. Briefly, the MRI properties of magnetic particles vary greatly depending on the coating thickness over the magnetic cores and their aggregated state, even though the amount of magnetic material remains constant. Additionally, MRI contrast agents have vastly different properties based on water solubility. We will synthesize different classes of biopolymer coated metallic core particles whose coating will be able to be enzymatically cleaved and removed. The biopolymer coatings will be biocompatible, yet highly resistant to passive degradation in the cells. Furthermore, for some particles, the metallic core will be able to dissolve. Dynamic manipulations of these phenomena will result in large enhancements of relaxivity and hence, of the MRI signal. Computer simulations predicting relaxivity changes as a function of coating thickness will guide the nanoparticle fabrication. We will then test whether enzyme-mediated removal of the biopolymer coating indeed results in modulation of the MRI properties. Simulations predict a 10-fold increase in r2, a 4-fold increase in r2* for iron oxide based particles, and a 50-fold increase in r1 for manganese based particles. These new classes of particles will form the technological core for non-invasive visualization of stem cell differentiation in intact organisms by MRI and can be further generalized to monitoring gene expression. Upon internalization into cells, nano- and microparticles are sequestered in endosomes and lysosomes. While it has been known that dextran coated magnetic particles slowly degrade within these structures, no study has yet purposely harnessed this phenomenon. The innovation of this proposed work is that we are utilizing the chemical environment within these subcellular structures as a medium for enzymatic reactions that will modulate water accessibility to the metallic cores, and in some cases, to dissolve them. Future implementation of these particles will utilize reporter enzymes to react with particles, engineered as transgenes, which will be expressed during the transition from stem cell to mature cell. So, rather than particles slowly degrading over several weeks, we will force the decomposition of these constructs to occur within hours once triggered, thus providing a relatively rapid, non-invasive, high resolution, three dimensional readout of stem cell differentiation.