MRI Contrast Agents for In vivo Monitoring of Stem Cell Differentiation

描述(申请人提供)：干细胞有望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。基于磁粒子的MRI细胞跟踪技术已被用于检测活体细胞的迁移，但目前尚不能检测细胞分化。这是因为目前用于细胞跟踪的磁性颗粒无论是在干细胞内还是在成熟细胞内，都会产生相同的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包裹的金属纳米颗粒，这将使磁共振成像能够在体内非侵入性地检测干细胞分化。这些新粒子的基本原理是这些粒子基于酶引发的生物聚合物涂层的修饰而实现摩尔弛豫度的大幅增强的能力。简而言之，即使磁性材料的量保持不变，磁性颗粒的MRI特性也会根据磁芯上的涂层厚度和它们的聚集状态而变化很大。此外，MRI造影剂基于水的溶解度有很大的不同性质。我们将合成不同类别的生物聚合物涂层金属核心颗粒，其涂层将能够被酶切割和去除。生物聚合物涂层将是生物兼容的，但高度抵抗细胞中的被动降解。此外，对于某些粒子，金属核将能够溶解。对这些现象的动态处理将导致弛豫度的大幅增强，从而导致MRI信号的增强。预测弛豫度随涂层厚度变化的计算机模拟将指导纳米颗粒的制备。然后，我们将测试酶介导的生物聚合物涂层的去除是否真的会导致MRI特性的调节。模拟预测R2将增加10倍，铁氧化物粒子的R2*将增加4倍，而锰基粒子的R2将增加50倍。这些新型颗粒将形成核磁共振成像非侵入性显示完整生物体中干细胞分化的技术核心，并可进一步推广到监测基因表达。在内化到细胞内后，纳米和微米颗粒被隔离在内质体和溶酶体中。虽然已经知道葡聚糖包覆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一现象。这项拟议工作的创新之处在于，我们利用这些亚细胞结构中的化学环境作为酶反应的媒介，这些反应将调节水对金属核心的可及性，在某些情况下，还将使它们溶解。未来这些颗粒的实现将利用报告酶来与转基因颗粒反应， 它将在干细胞向成熟细胞的转变过程中表达。因此，我们将迫使这些结构在触发后几个小时内分解，而不是粒子在几周内缓慢降解，从而提供相对快速、非侵入性、高分辨率的干细胞分化三维读数。

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.