Pre-clinical evaluation of Magnetically labeled Cells for Cellular MRI

此前，我们将常用的硫酸鱼精蛋白(Pro)与超顺磁性葡聚糖包被的纳米氧化铁(SPIO)铁氧化物(FE)结合在一起，形成了一种可用于对干细胞和其他哺乳动物细胞进行磁性标记的复合体。细胞通过巨噬细胞吞噬来摄取FEPro复合体。通过细胞核磁共振早期检测体内标记细胞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。我们以前修改了目前的标记方法，使用低剂量的Pro，并在产生任何FePro络合物之前直接向细胞中添加Fe和Pro。为了验证这一新方法，标记了人T细胞、人造血干细胞(HHSC)和人骨髓基质细胞(HMSC)。用100g/mlFe和3g/mlPro标记4小时，可以非常有效地标记这些细胞，而不会影响它们的活性和功能。然而，阿魏酸铁被从市场上撤下，阿魏酸甘油(F)是FDA批准的用于治疗慢性肾脏疾病缺铁性贫血的纳米氧化铁颗粒，于2009年推出。阿魏酸甘油酯(F)是一种直径为17-31 nm的纳米粒子，以6 nm的氧化铁晶体为核心，也被用于实验和临床试验中，通过MRI作为巨噬细胞显像剂和血池试剂。在2009年末，我们表征了F和P纳米络合物在水中的物理化学性质。在水中，FS Zeta电位为-55 mV，而鱼精蛋白(P)为7.2 mV。这两个纳米复合体通过静电相互作用形成了大于400 nm的FP颗粒；然而，我们不能用两种药物的任何比例来标记细胞。添加硫酸肝素(H)使我们能够在培养中标记癌细胞系。基于肝素的纳米络合物已被证明可以与各种阳离子分子自组装，并已被用于药物输送、组织工程或延长试剂的循环半衰期。虽然将肝素添加到PF中以促进细胞内的内体结合是违反直觉的，但添加肝素会导致HPF纳米复合体的形成，这些纳米复合体被细胞内吞。HPF的透射电子显微镜显示，这些纳米复合体呈球形，中间的HP被F包围。将干细胞和T细胞在HFP纳米复合体中在无血清介质中孵育2小时，然后在完全介质中进行细胞标记。HPF标记不影响细胞的活力、增殖能力、凋亡率、活化、表型表面标志的表达或分化能力。将HPF标记的骨髓间充质干细胞植入大鼠脑内，3T时的磁共振成像显示，与周围实质相比，大鼠脑内可检测到1000个细胞，T2*减少了50%。HPF标记技术已经规模化，NIH细胞处理部门的cGMP设施能够在生物工厂标记BMSCs，而标记的低温保存细胞产品的BMSC功能或活性不会改变。HPF标记方法有助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潜能干细胞是否可以分化为荧光标记的sigma-2((2))受体探针，使用SW120评估了人类干细胞(SC)中受体的表达，包括：骨髓基质细胞、神经前体细胞、羊水细胞、造血细胞和胚胎干细胞。Sigma-2(&#963；(2))受体是实体肿瘤增殖状态的潜在生物标志物。利用N-取代-9-氮杂二环3.3.1-农-3和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定合成探针。干细胞与AT荧光标记的SW120分子孵育一小段时间，我们能够证明快速结合和整合到SC。我们评估了SW120和增殖标志物5-乙炔-2‘-脱氧尿苷(EDU)的强度与细胞传代数和多效性的关系，结果表明：(2)增殖干细胞中的受体密度明显高于谱系受限的细胞类型。干细胞中受体的细胞内化与受体介导的内吞作用是一致的，共聚焦显微镜显示SW120与所有成像的SC中的溶酶体的荧光标记共同定位。这些结果表明，Sigma-2受体可用于体内监测干细胞分化，并计划开发一种基于SW120骨架的分子成像探针，用于通过MRI或正电子发射断层扫描无创追踪干细胞分化。

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.