MRI Contrast Agents for In vivo Monitoring of Stem Cell Differentiation

描述（由申请人提供）：干细胞治疗前景的关键是非侵入性检测细胞迁移和分化的方法。使用磁性颗粒的基于MRI的细胞追踪已被用于检测体内细胞迁移，然而，其目前不能检测细胞分化。这是因为目前用于细胞跟踪的磁性颗粒无论是在干细胞还是成熟细胞内都能产生相同的MRI对比度。在这里，我们建议设计，合成和表征新型的生物聚合物封装的金属纳米颗粒，这将使使用MRI非侵入性检测干细胞分化，在体内。这些新颗粒的基本原理是基于生物聚合物涂层的酶触发改性，这些颗粒实现摩尔弛豫率大幅增强的能力。简而言之，磁性颗粒的MRI性质根据磁芯上的涂层厚度及其聚集状态而变化很大，即使磁性材料的量保持恒定。此外，MRI造影剂具有基于水溶性的极大不同的性质。我们将合成不同种类的生物聚合物涂层的金属核心颗粒，其涂层将能够被酶促裂解和去除。生物聚合物涂层将是生物相容的，但对细胞中的被动降解具有高度抗性。此外，对于某些颗粒，金属核将能够溶解。这些现象的动态操纵将导致弛豫率的大幅增强，从而导致MRI信号的大幅增强。计算机模拟预测弛豫率变化作为涂层厚度的函数将指导纳米颗粒的制造。然后，我们将测试是否酶介导的生物聚合物涂层的去除确实导致调制的MRI性能。模拟预测r2增加10倍，对于铁氧化物基颗粒r2* 增加4倍，对于锰基颗粒r1增加50倍。这些新的颗粒类别将形成通过MRI对完整生物体中的干细胞分化进行非侵入性可视化的技术核心，并且可以进一步推广到监测基因表达。在内化到细胞中后，纳米颗粒和微米颗粒被隔离在内体和溶酶体中。虽然已知葡聚糖涂层的磁性颗粒在这些结构中缓慢降解，但还没有研究有目的地利用这种现象。这项工作的创新之处在于，我们正在利用这些亚细胞结构内的化学环境作为酶反应的介质，这将调节水对金属核心的可及性，并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作为转基因工程的颗粒反应， 其将在从干细胞到成熟细胞的转变期间表达。因此，不是颗粒在几周内缓慢降解，而是一旦触发，我们将迫使这些构建体在数小时内发生分解，从而提供相对快速，非侵入性，高分辨率，干细胞分化的三维读数。

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.