Pre-clinical evaluation of Magnetically labeled Cells for Cellular MRI

以前，我们将常用的鱼精蛋白硫酸盐（Pro）与超顺磁性葡聚糖包覆的氧化铁纳米颗粒（SPIO）铁氧化物（FE）结合在一起，形成了一种复合物，可用于磁性标记干细胞和其他哺乳动物细胞。细胞通过巨噬作用吸收FEPro复合物。通过细胞MRI对体内标记细胞的早期检测需要开发新的脉冲序列或图像处理，以提高对组织中少量氧化铁标记细胞的敏感性。我们之前修改了目前的标记方法，使用低剂量的Pro，并在生成任何FePro复合物之前将Fe和Pro直接添加到细胞中。为了验证这种新方法，我们对人t细胞、人造血干细胞（hHSC）、人骨髓基质细胞（hMSC）进行了标记。使用100g/ml Fe和3g/ml Pro标记4小时，可以非常有效地标记这些细胞，而不会损害它们的活力和功能能力。然而，阿魏氧化物被撤出市场，阿魏木糖醇(F)是一种经FDA批准用于治疗慢性肾病缺铁性贫血的氧化铁纳米颗粒，于2009年推出。阿鲁莫xytol (F)是一种直径为17-31 nm的纳米颗粒，其核心为6 nm的氧化铁晶体，在实验和临床试验中也被用作巨噬细胞显像剂和MRI血池剂。2009年末，我们对水中氟和磷纳米配合物的理化性质进行了表征。f的zeta电位为-55mV，而鱼精蛋白(P)在水中的zeta电位为7.2mV。两种纳米配合物通过静电相互作用形成大于400nm的FP粒子；然而，我们无法用两种药物的任何比例标记细胞。添加硫酸肝素(H)使我们能够在培养中标记癌细胞系。以肝素为基础的纳米复合物已被证明可以与多种阳离子分子自组装，并已被用于药物输送、组织工程或延长药物的循环半衰期。虽然将肝素添加到PF中以促进细胞内体结合是违反直觉的，但肝素的添加导致HPF纳米复合物的形成，并被细胞内吞。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。HPF标记技术已经扩大，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标记方法应便于临床试验中移植细胞的MRI监测。为了确定是否有可能使用小分子来确定多能干细胞是否分化荧光标记的sigma-2（σ(2)）受体探针，我们使用SW120来评估&#963；(2)受体在人干细胞（SC）中的表达，包括：骨髓基质、神经祖细胞、羊水、造血和胚胎干细胞。sigma-2（σ(2)）受体是实体瘤增殖状态的潜在生物标志物。利用n-取代-9-阿扎比环3.3.1nonan-3&#945；-氨基甲酸酯类似物设计并实现了用于实验性癌症诊断和治疗的特异性合成探针。干细胞与荧光标记的SW120分子孵育短时间，我们能够证明快速结合并结合到SC中。我们评估了SW120的强度和增殖标记物5-乙基-2'-脱氧尿苷（EdU）相对于细胞传代数和多能性，并显示增殖干细胞中相对于谱系限制细胞类型的受体密度显着更高。干细胞中&#963；(2)受体的细胞内化与受体介导的内吞作用一致，共聚焦显微镜显示所有SC图像中SW120特异性与溶酶体荧光标记共定位。这些结果表明，sigma -2受体可用于监测体内干细胞分化，并且正在计划开发基于SW120骨干的分子成像探针，用于通过MRI或正电子发射断层扫描无创跟踪干细胞分化。

Previously we combined commonly used Protamine Sulfate (Pro) with superparamagnetic dextran coated iron oxide nanoparticle (SPIO) Ferumoxides (FE), a complex is formed that can be used to magnetically label stem cells and other mammalian cells. Cells take up FEPro complexes by macropinocytosis. Early detection of labeled cells in vivo by cellular MRI requires the development of novel pulse sequences or image processing to improve the sensitivity to low numbers of iron oxide labeled cells in tissues. We previously revise the current labeling method by using low dose of Pro and adding Fe and Pro directly to the cells before generating any FePro complexes. To validate this new approach human T-cells, human hematopoietic stem cells (hHSC), human bone marrow stromal cells (hMSC) were labeled. Labeling for 4 hours using 100g/ml of Fe and 3g/ml of Pro resulted in very efficient labeling of these cells, without impairing their viability and functional capability. However, ferumoxides were taken off the market place and ferumoxytol (F) an FDA approved iron oxide nanoparticle used to treat iron deficiency anemia for chronic kidney disease was introduced in 2009. Ferumoxytol (F) is a nanoparticle of 17-31 nm in diameter with the 6 nm iron oxide crystals core and has been also used in experimental and clinical trials as a macrophage imaging agent as well as blood pool agent with MRI. In late 2009, we characterized the physiochemical properties of F and P nanocomplexes in water. Fs zeta potential is -55mV while protamine (P) is 7.2mV in water. The two nanocomplexes form greater than 400 nm particles of FP via electrostatic interactions; however we could not label cells with any ratio of the two drugs. The addition of heparin sulfate (H) resulted in our ability to label cancer cell lines in culture. Heparin-based nanocomplexes have been shown to self-assemble with a variety of cationic molecules and have been used for drug delivery, tissue engineering or for prolonging circulating half-life of an agent. Although it was counterintuitive to add heparin to PF to facilitate endosomal incorporation in cells, the addition of heparin resulted in the formation of HPF nanocomplexes that were endocytosed by 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. The HPF labeling technique has been scaledup, 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 labeling method should facilitate the monitoring by MRI of transplanted cells in clinical trials. In order to determine if it were possible to use a small molecule to determine if multipotential stem cells differentiated a fluorescently labeled sigma-2 (σ(2)) receptor probe, SW120 was used to evaluate σ(2) receptor expression in human stem cells (SC), including: bone marrow stromal, neural progenitor, amniotic fluid, hematopoietic, and embryonic stem cells. The sigma-2 (σ(2)) receptor is a potential biomarker of proliferative status of solid tumors. Specific synthetic probes using N-substituted-9-azabicyclo 3.3.1nonan-3α-yl carbamate analogs have been designed and implemented for experimental cancer diagnosis and therapy. Stem cells were incubated with at fluorescently tagged SW120 molecule for short periods of time and we were able to demonstrate rapid binding and incorporation into the SC. We evaluated the intensity of SW120 and a proliferation marker 5-ethynyl-2'-deoxyuridine (EdU) relative to cell passage number and multi-potency and showed that significantly higher σ(2) receptor density among proliferating stem cells relative to lineage-restricted cell types. Cellular internalization of the σ(2) receptor in stem cells was consistent with receptor-mediated endocytosis and confocal microscopy indicated SW120 specific co-localization with a fluorescent marker of lysosomes in all SC imaged. These results suggested that the sigma -2 receptor could be used to monitor stem cell differentiation in vivo and plans are underway to develop a molecular imaging probe based on the SW120 backbone for noninvasive tracking of stem cell differentiation by MRI or positron emission tomography.