Pre-clinical evaluation of Magnetically labeled Cells for Cellular MRI

在此之前，我们证明了阿魏木糖醇(F)与鱼精蛋白(P)和肝素(H)结合可以产生一种可用于标记细胞的自组装纳米复合物。HFP的透射电镜显示，这些纳米复合物呈球形，中间的HP被f包围，干细胞和t细胞在HFP纳米复合物中无血清培养液中孵育2小时，然后完全培养液进行细胞标记。HPF标记不影响细胞活力、增殖能力、凋亡率、活化、表型表面标记表达或分化能力。HPF标记的BMSC植入大鼠脑后3T MRI显示，与周围实质相比，大鼠脑内T2*减少50%，能够检测到1000个细胞。对小鼠脑内给药hpf标记的NSCs进行了临床前安全性/毒性研究，结果显示没有明显的临床或行为改变，没有神经元或全身毒性，也没有在肝脏或脾脏中异常积累铁。HPF标记技术已经扩大了规模，NIH细胞处理部门的cGMP设施能够在生物工厂中标记骨髓间充质干细胞，而标记的冷冻保存细胞产品的骨髓间充质干细胞功能或活力没有变化。HPF方法也被用于标记表达胞嘧啶脱氨酶的基因工程神经干细胞（NSC），作为治疗复发性胶质母细胞瘤患者的正在进行的临床试验的一部分。HPF标记的NSC将直接植入手术切除视力周围的大脑深处，以便它们能够迁移到残留肿瘤或卫星转移区域。MRI监测了30天内HPF标记的NSC在大脑中的迁移。在希望之城进行的初步研究报告说，在植入标记细胞后基本上没有不良事件。我们最近优化了HPF标记方法，通过改变将三种药物添加到细胞中以FHP的顺序，导致与HPF标记的细胞相比，BMSC和NSC的细胞内铁含量显着增加。我们还报道了优化不同比例的H、P和F组分的物理化学特性，以及混合顺序，产生不同水动力尺寸的nc。NC的大小取决于药物在介质中混合的顺序。HPF和FHP的电子显微镜显示，F位于球形HP配合物的表面。与相同f浓度的HPF NCs相比，用FHP NCs培养的人干细胞每个细胞的铁浓度明显更高。这些结果表明，FHP可用于临床转化研究中的干细胞标记。

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.