Pre-clinical evaluation of Magnetically labeled Cells for Cellular MRI

之前，我们证明了通过将ferumoxytol（F）与鱼精蛋白（P）和肝素（H）组合，产生了可用于标记细胞的自组装纳米复合物。透射电子显微镜观察表明，这些纳米复合物呈球形，HP位于中心，周围环绕着F。将干细胞和T细胞在无血清培养基中的HFP纳米复合物中孵育2小时，然后完全培养基导致细胞标记。HPF标记不损害细胞活力、增殖能力、凋亡率、活化、表型表面标志物表达或分化能力。植入大鼠脑中的HPF标记的BMSC在3 T下的MRI证明了检测1000个细胞的能力，与周围实质相比，大鼠脑中的T2* 降低50%。还在小鼠中进行了脑内施用HPF标记的NSC的临床前安全性/毒性研究，并且证明没有显著的临床或行为变化，没有神经元或全身毒性，并且肝脏或脾脏中没有铁的异常积累。HPF标记技术已经扩大规模，NIH细胞处理部门cGMP设施能够在生物工厂中标记BMSC，而标记的冷冻保存细胞产品的BMSC功能或活力没有变化。HPF方法还用于标记表达胞嘧啶脱氨酶的遗传工程神经干细胞（NSC），作为正在进行的治疗复发性胶质母细胞瘤患者的临床试验的一部分。 将HPF标记的NSC直接植入手术切除视野周围的脑深部，以便它们可以迁移到残留肿瘤或卫星转移的区域。 MRI监测了30天内HPF标记的NSC在脑中的迁移。在City of Hope进行的初步研究报告称，植入标记细胞后基本没有不良事件。 我们最近优化了HPF标记方法，通过改变三种药物加入细胞的顺序，FHP导致BMSC和NSC中细胞内铁含量的显着增加相比，HPF标记的细胞。 我们还报告的物理化学特性优化的H，P，和F组分在不同的比例，和混合序列，产生不同的流体动力学大小的NC。NC大小取决于药物在介质中混合的顺序。HPF或FHP的电子显微镜显示F位于球形HP复合物的表面。与具有相同浓度的F的HPF NC相比，与FHP NC孵育的人干细胞导致每个细胞的铁浓度显著更高。这些结果表明，FHP可能是有用的标记干细胞在临床上的翻译研究。

Previously, we demonstrated that by combining ferumoxytol (F) with protamine (P) and heparin (H) resulted in a self assembling nanocomplex that could be used to label cells. Transmission electron microscopy of HPF revealed that these nanocomplexes were spheroid shaped with the HP in center surrounded by F. Incubating stem cells and T-cells in HFP nanocomplexes in serum free media for 2 hours followed by complete media resulted in cell labeling. HPF labeling did not impair the cells viability, proliferative capacity, apoptotic rate, activation, phenotypic surface marker expression, or capacity to differentiate. MRI at 3T of HPF labeled BMSC implanted in the rat brain demonstrated the ability to detect 1000 cells with a 50% decrease in T2* in the rat brain compared to the surrounding parenchyma. Pre-clinical safety/toxicity studies of intracerebrally administrated HPF-labeled NSCs in mice were also performed, and demonstrated no significant clinical or behavioral changes, no neuronal or systemic toxicities, and no abnormal accumulation of iron in the liver or spleen. The HPF labeling technique has been scaled up, and the NIH Cell Processing Section cGMP facility was able to label BMSCs in biofactories with no changes in BMSC function or viability of the labeled cryopreserved cell product. The HPF method was also used to label genetically engineered neural stem cells (NSC) that express cytosine deaminase as part of an ongoing clinical trial to treat patients with recurrent glioblastoma. The HPF labeled NSC are to be directly implanted deep into brain around the periphery of the surgical resection sight in order that they can migrate to areas of residual tumors or satellite metastasis. MRI monitored the migration of the HPF labeled NSC in the brain over 30 days. Initial studies performed at City of Hope report that there was essential no adverse events following the implantation of labeled cells. We have recently optimized the HPF labeling approach by changing the order that the three drugs are added to cells to FHP resulted in significant increase in the intracellular iron content in BMSC and NSC as compared to HPF labeled cells. We also report on the physicochemical characteristics for optimizing the H, P, and F components in different ratios, and mixing sequences, producing NCs that varied in hydrodynamic size. NC size depended on the order in which drugs were mixed in media. Electron microscopy of HPF or FHP showed that F was located on the surface of spheroidal shaped HP complexes. Human stem cells incubated with FHP NCs resulted in a significantly greater iron concentration per cell compared to that found in HPF NCs with the same concentration of F. These results indicate that FHP could be useful for labeling stem cells in translational studies in the clinic.