叶酸受体介导的肿瘤靶向超顺磁性纳米胶束的制备及体外实验

目的探讨叶酸受体介导的两亲聚合物纳米胶束对人肝癌Bel 7402细胞的靶向性，及利用MR仪对其进行监测的可行性。方法制备由叶酸修饰的、载有超顺磁性氧化铁（SPIO）及抗癌药物表阿霉素（DOX）的纳米胶束，将叶酸靶向及非叶酸靶向纳米胶束分别与人肝癌Bel 7402细胞共孵育1h，进行普鲁士蓝染色和流式细胞术观察Bel 7402细胞对叶酸靶向及非叶酸靶向纳米胶束的吸收情况，并体外MRI观察T2WI信号强度变化。结果叶酸靶向纳米胶束与Bel 7402细胞共孵育后普鲁士蓝染色显示细胞内大量铁存在；非叶酸靶向纳米胶束普鲁士蓝染色显示细胞内铁浓度极低。流式细胞术显示叶酸靶向组及非叶酸靶向组的平均荧光强度分别为117．88和46．33，叶酸靶向组约为非叶酸靶向组的2．5倍。体外MRI显示叶酸靶向纳米胶束与Bel 7402细胞共孵育后在T2WI上信号明显降低（SPIO浓度为5、10、20、40和80μg／ml时，信号变化率中位数分别为-5．02％、-23．58％、-45．89％、-70．34％和-92．41％），而非叶酸靶向组在T2WI上信号无明显降低（SPIO浓度为5、10、20、40和80μg／ml时信号变化率中位数分别为-3．77％、-2．16％、-2．18％、-2．74％和-19．77％）。体外竞争抑制实验普鲁士蓝染色显示细胞内铁浓度极低。结论以叶酸修饰的生物可降解聚合物纳米胶束对人肝癌细胞Bel 7402有较好的靶向性，使用临床型MR仪可对其进行监测。     

超顺磁性氧化铁纳米颗粒标记兔骨髓间充质干细胞的孵育时间优选

目的探讨超顺磁性氧化铁(SPIO)纳米颗粒标记兔骨髓间充质干细胞(MSCs)的最佳孵育时间。方法分离培养兔骨髓单个核细胞5×105/8ml共4瓶,SPIO分别以10μl、20μl、60μl浓度加入其中3瓶标记,另1瓶为未标记细胞。均在37°C,5%CO2条件下孵化过夜,6h及以后每隔6h直至标记后7d分别经荧光显微镜观察标记细胞的形态学改变和铁颗粒结合率,并与未标记组细胞进行对照。结果3组均显示细胞内吞铁颗粒随着孵育时间的延长而增加,标记浓度越高则细胞内吞铁颗粒越多;10μl组标记后18~24h标记率可达到96%~100%,且细胞的形态与增殖分化能力正常,与未标记细胞组基本一致,孵育时间<18h则标记率<90%;20μl组标记后12h细胞标记率即达到100%,标记后3~7d,细胞分裂增殖不明显;60μl组标记后6h标记率即达到100%,但以后细胞的分化增殖能力明显受限。结论安全标记浓度下磁性标记干细胞的最佳孵育时间为18~24h。标记率与标记浓度和标记后孵育时间呈正相关。     

不同浓度菲立磁对大鼠间充质干细胞标记效率和细胞活力的影响

目的探讨不同浓度菲立磁标记大鼠骨髓间充质干细胞（MSCs）的磁标记效率及对细胞生长活力的影响，寻找最佳标记浓度。材料与方法菲立磁与多聚左旋赖氨酸（PLL）混合制备菲立磁-PLL复合物。将不同浓度的菲立磁-PLL复合物与培养基混合（铁的终浓度分别为150μg／ml、100μg／ml、50μg／ml、25μg／ml、10μg／ml和5μg／ml），加入MSCs孵育过夜。分别于标记后1天、1周、2周、3周、4周行铁染色、铁含量测定及细胞活力检测。结果菲立磁-PLL标记组细胞铁含量显著高于单纯菲立磁标记组（P＜0．05），25μg／ml浓度组细胞铁含量显著高于10μg／ml及其以下浓度组（P＜0．05），但与50μg／ml及其以上浓度组差异无统计学意义（P＞0．05）。台盼蓝排除试验显示，100μg／ml及其以下浓度组细胞活力与对照组之间差异无统计学意义（P＞0．05）。结论以25μg/ml铁浓度标记干细胞不仅标记效率高，而且对细胞活力无明显影响．为菲立磁标记MSCs的最佳浓度。     

超顺磁性氧化铁标记鼠骨髓间充质干细胞的体外MR成像研究

目的: 探讨不同浓度超顺磁性氧化铁(SPIO)颗粒标记鼠骨髓间充质干细胞(MSCs)的标记率和对细胞活力的影响,以及MR成像显示磁标记干细胞的可行性.材料和方法: 将不同浓度的SPIO-PLL复合物与培养基混合,行普鲁士蓝染色观察细胞内铁和检测细胞活力.应用1.5T MR仪,以T1WI和T2WI行磁标记干细胞成像.结果: SPIO可有效标记MSCs,标记后的铁颗粒位于细胞质内.SPIO标记的MSCs可引起T2WI信号降低,50、100、150较25μg Fe /ml的信号强度降低明显.结论: SPIO可以简便标记MSCs,并在适当浓度下对细胞活力没有影响,此技术为干细胞移植的MR活体内示踪奠定基础.     

超顺磁性氧化铁标记小鼠淋巴细胞MR成像特性探讨

目的 探讨超顺磁性氧化铁(SP IO)标记小鼠脾脏淋巴细胞及其体外MR成像的可行性.方法 取5只小鼠脾脏淋巴细胞,采用100、50、25、15、10、5μg/ml的SPIO联合3μg/ml的多聚赖氨酸(PLL)标记淋巴细胞,确定最佳标记浓度.普鲁士蓝染色鉴定细胞内铁.取30份新鲜、标记、未标记淋巴细胞行台盼蓝染色检测细胞存活率的变化.用对照组及2×106、5×106和10×106个/ml的标记后淋巴细胞接种于琼脂糖凝胶中行3.0 T MR T2 WI、T2+WI及磁敏感加权成像(SWI)序列扫描,相同细胞浓度不同序列及相同序列不同细胞浓度时各取21个感兴趣区测量MR信号值.采用组间独立样本t检验比较细胞存活率;单因素方差分析比较MR信号值差异.结果 SPIO联合PLL成功标记小鼠淋巴细胞,最佳SPIO浓度为5μg/ml.普鲁士蓝染色显示细胞内存在蓝染铁颗粒,阳性率为(93.6±2.1)％.30份台盼蓝染色细胞样本,新鲜分离的淋巴细胞存活率为(94.8±3.1)％,细胞培养6h后,标记组和未标记组淋巴细胞存活率分别为(88.7±2.7)％和(88.9±3.2)％.标记组同未标记组细胞间存活率差异无统计学意义(t =0.281,P＞0.05),但标记组、未标记组存活率同培养前相比,其下降均有统计学意义(t值分别为8.125、7.253,P值均＜0.05).相同浓度细胞,T2WI信号降低最弱,SWI信号降低最明显.结论 SPIO联合PLL能有效标记小鼠淋巴细胞,且不影响其存活率,标记细胞体外3.0T MR成像可行,以SWI序列最敏感.     

两种新型竹红菌乙素衍生物在人胃腺癌BGC-823细胞中吸收规律的研究

目的探讨两种新型光敏剂5-氨基-1-戊磺酸竹红菌乙素衍生物（PENSHB）和15位脱乙酰基13位3-氨基-1-丙磺酸竹红菌乙素衍生物（DPROHB）在人胃腺癌BGC-823细胞中的吸收规律。方法以浓度为4μM的PENSHB、DPROHB对人胃腺癌BGC-823细胞分别孵育0、0.5、1、2、4和8 h,用荧光分析法测定细胞内光敏剂含量,通过浓度-荧光强度标准曲线换算成光敏剂浓度,绘制每种光敏剂的孵育时间-细胞含量的关系曲线。以浓度为0.5、1、2、4和8μM的两种光敏剂分别孵育BGC-823细胞4 h后,测定细胞内荧光强度,绘制光敏剂的孵育浓度-细胞含量关系曲线。结果 BGC-823细胞对PENSHB和DPROHB的吸收含量随孵育时间的延长而增加,两者变化趋势相近,孵育1 h内细胞吸两种新型光敏剂收含量增加较快,4 h达到平台。在本实验的浓度范围内PENSHB和DPROHB的细胞吸收量随孵育浓度的增高而增加,基本呈线性关系,上升趋势相似。两者在细胞内的吸收含量依赖孵育时间及孵育浓度。结论两种新型竹红菌乙素衍生物PENSHB和DPROHB在提高水溶性及光动力效应的同时,细胞吸收特性良好,是有潜在应用前景的新型光敏剂。     

超小超顺磁性氧化铁微粒对神经干细胞生物学活性的影响

目的应用超小超顺磁性氧化铁微粒Sinerem标记大鼠骨髓源性神经干细胞（NSCs），探讨Sinerem的安全性及合适的标记浓度。方法分离、培养大鼠骨髓源性NSCs。制备Sinerem多聚赖氨酸复合物，以不同浓度Sinerem对细胞进行标记。普鲁士蓝染色和电镜观察细胞内铁，CCK-8法检测细胞生长增殖情况，AnnexinV—PI法检测细胞凋亡及死亡情况。结果普鲁士蓝染色显示细胞质内大量铁颗粒存在，标记率在99％以上。电镜观察见Sinerem标记干细胞内含纳米铁颗粒。CCK-8法检测结果表明，25～1500μg/ml不同浓度范围的Sinerem对细胞增殖的影响差异无统计学意义。AnnexinV—PI检测结果显示，Sinerem在25～200μg/ml范围内的不同浓度对细胞凋亡和死亡的影响差异无统计学意义。结论超小超顺磁性氧化铁微粒Sinerem可以有效标记大鼠骨髓源性NSCs，可以应用25～200μg／ml浓度范围的Sinerem标记细胞。     

心脏磁共振在体示踪移植细胞的可行性研究

目的：寻找一种能够对移植细胞进行在体示踪的标记方法,为移植细胞存留、迁移提供重要观察手段。方法：从中华小型猪髂骨处抽取骨髓,体外培养扩增骨髓间充质干细胞（MSCs）。将SPIO和MSCs共同孵育培养36h。普鲁士蓝染色评价细胞的标记效率;通过MTT比色实验评价SPIO对细胞生长能力的影响;台盼蓝染色检验标记后细胞的活性;使用Costar Transwell方法评价铁离子对细胞迁移能力的影响;用细胞分化诱导液培养标记后的细胞评价其向成脂肪细胞和成骨细胞的分化能力。在体内实验中将SPIO标记或未标记的自体MSCs注射到心肌内,通过心脏磁共振检查对移植细胞进行在体示踪观察,取材动物心脏行病理检查观察移植细胞的存活、存留。结果：MSCs经铁离子标记后普鲁士蓝染色阳性率在98%以上,可见蓝色颗粒位于细胞浆内,标记细胞电镜切片可见高密度铁颗粒位于细胞浆内。随着培养液中SPIO浓度的增加细胞增殖能力没有明显改变;标记后98%的细胞保持活性;SPIO标记后的细胞保持原有的形态,可继续培养、传代;SDF-1和VEGF诱导的迁移实验发现标记细胞迁移能力没有降低;铁离子标记后细胞仍可向成脂肪细胞和成骨细胞分化。注射到心肌内的SPIO标记的MSCs可通过心脏磁共振检查进行在体示踪,动态观察显示SPIO标记细胞在磁共振图像上表现为低信号,并且在移植后4周仍可成像。病理学检查可以看到移植细胞呈普鲁士蓝染色阳性,并和影像学有很好的一致性。结论：临床使用的SPIO磁共振对比剂可以安全、有效地标记MSCs,心脏磁共振检查可以实现SPIO标记的移植细胞的在体示踪。     

SPIO标记间充质干细胞3TMR成像

目的：探讨小容积动物线圈3.0T临床磁共振扫描仪体外示踪磁标记间充质干细胞的可行性。方法：用GFP转染法将含有不同剂量铁（56ug／ml和560ug／m1）的超顺磁氧化铁（SPIO）标记间充质干细胞。细胞内铁的含量利用原子吸收谱来测量。磁共振扫描和显微镜可以观察培养试管中的磁标记间充质干细胞。     

体外标记脂肪源性干细胞移植治疗缺血性脑损伤的MRI示踪成像研究

目的 采用多聚左旋赖氨酸(PLL)与超顺磁氧化铁纳米微粒(SPIO)的复合物对脂肪源性干细胞(AD-SCs)体外进行标记,观察ADSCs脑内移植治疗大鼠缺血性脑损伤后的增殖和迁移情况.材科与方法显微镜下直接结扎大脑中动脉方法制备大鼠缺血性脑损伤模型36只,随机分成缺血对照组(12只)、磁性标记ADSCs移植组(12只)和未磁性标记ADSCs移植组(12只),采用立体定向方法脑内移植.对移植后大鼠的神经系统行为和运动功能进行评估,免疫组织化学染色观察ADSCs的存活及分化情况,并用MRI在体观察ADSCs的存活和分布.结果 免疫组织化学检查显示脑内移植后部分标记细胞表达神经元特异性烯醇化酶(NSE)和血管内皮细胞标记物CD31,移植后3周神经系统行为学评分显示移植组动物明显改善,ADSCs脑内移植后3周MR T2WI、GRET2*WI订显示移植区低信号改变并通过胼胝体向病灶迁移.结论 移植ADSCs可以有效地促进缺血性大鼠神经行为功能的恢复,SPIO和PLL复合物标记方法能够评价细胞移植后的细胞迁移.     

Transferrin receptor upregulation: in vitro labeling of rat mesenchymal stem cells with superparamagnetic iron oxide

PURPOSE: To prospectively evaluate the influence of superparamagnetic iron oxide (SPIO) or ultrasmall SPIO (USPIO) particles on the surface epitope pattern of adult mesenchymal stem cells (MSCs) by regulating the expression of transferrin receptor and to prospectively evaluate the influence of transfection agents (TAs) on the uptake of SPIO or USPIO particles in MSCs. MATERIALS AND METHODS: The study was approved by the institutional animal care committee of the University of Tübingen. MSCs were isolated from the bone marrow of four rats. To obtain highly homogeneous MSC populations, MSCs from one rat were single-cell cloned. One MSC clone was characterized and selected for the labeling experiments. The MSCs, which were characterized with flow cytometry and in vitro differentiation, were labeled with 200 microg/mL SPIO or USPIO or with 60 microg/mL SPIO or USPIO in combination with TAs. Aggregations of labeled cells were accommodated inside a defined volume in an agar gel matrix. Magnetic resonance (MR) imaging was performed to measure SPIO- or USPIO-induced signal voids. Quantification of cellular total iron load (TIL) (intracellular iron plus iron coating the cellular surface), determination of cellular viability, and electron microscopy were also performed. RESULTS: Labeling of MSCs with SPIO or USPIO was feasible without affecting cell viability (91.1%-94.7%) or differentiation potential. For MR imaging, SPIO plus a TA was most effective, depicting 5000 cells with an average TIL of 76.5 pg per cell. SPIO or USPIO particles in combination with TAs coated the cellular surface but were not incorporated into cells. In nontransfected cells, SPIO or USPIO was taken up. MSCs labeled with SPIO or USPIO but without a TA showed enhanced expression of transferrin receptor, in contrary to both MSCs labeled with SPIO or USPIO and a TA and control cells. CONCLUSION: SPIO or USPIO labeling without TAs has an influence on gene expression of MSCs upregulating transferrin receptor. Furthermore, SPIO labeling with a TA will coat the cellular surface.     

Physical and biological characterization of superparamagnetic iron oxide- and ultrasmall superparamagnetic iron oxide-labeled cells: a comparison

RATIONALE: Superparamagnetic iron-oxide particles are used frequently for cellular magnetic resonance imaging and in vivo cell tracking. The purpose of this study was to compare the labeling characteristics and efficiency as well as toxicity of superparamagnetic iron oxide (SPIO) and ultrasmall superparamagnetic iron oxide (USPIO) for 3 cell lines. METHODS: Using human fibroblasts, immortalized rat progenitor cells and HEP-G2-hepatoma cells, dose- and time-dependence of SPIO and USPIO uptake were evaluated. The amount of intracellular (U)SPIO was monitored over 2 weeks after incubation by T2-magnetic resonance relaxometry, ICP-mass-spectrometry, and histology. Transmission-electronmicroscopy was used to specify the intracellular localization of the endocytosed iron particles. Cell death-rate and proliferation-index were assessed as indicators of cell-toxicity. RESULT: For all cell lines, SPIO showed better uptake than USPIO, which was highest in HEP-G2 cells (110 +/- 2 pg Fe/cell). Cellular iron concentrations in progenitor cells and fibroblasts were 13 +/- 1pg Fe/cell and 7.2 +/- 0.3pg Fe/cell, respectively. For all cell lines T2-relaxation times in cell pellets were below detection threshold (<3 milliseconds) after 5 hours of incubation with SPIO (3.0 micromol Fe/mL growth medium) and continued to be near the detection for the next 6 days. For both particle types and all cell lines cellular iron oxide contents decreased after recultivation and surprisingly were found lower than in unlabeled control cells after 15 days. Viability and proliferation of (U)SPIO-labeled and unlabeled cells were not significantly different. CONCLUSIONS: The hematopoetic progenitor, mesenchymal fibroblast and epithelial HEP-G2 cell lines accumulated SPIO more efficiently than USPIO indicating SPIO to be better suited for cell labeling. However, the results indicate that there may be an induction of forced cellular iron elimination after incubation with (U)SPIO.     

Clinically applicable labeling of mammalian and stem cells by combining superparamagnetic iron oxides and transfection agents

PURPOSE: To label mammalian and stem cells by combining commercially available transfection agents (TAs) with superparamagnetic iron oxide (SPIO) magnetic resonance (MR) imaging contrast agents. MATERIALS AND METHODS: Three TAs were incubated with ferumoxides and MION-46L in cell culture medium at various concentrations. Human mesenchymal stem cells, mouse lymphocytes, rat oligodendrocyte progenitor CG-4 cells, and human cervical carcinoma cells were incubated 2-48 hours with 25 microg of iron per milliliter of combined TAs and SPIO. Cellular labeling was evaluated with T2 relaxometry, MR imaging of labeled cell suspensions, and Prussian blue staining for iron assessment. Proliferation and viability of mesenchymal stem cells and human cervical carcinoma cells labeled with a combination of TAs and ferumoxides were evaluated. RESULTS: When ferumoxides-TA or MION-46L-TA was used, intracytoplasmic particles stained with Prussian blue stain were detected for all cell lines with a labeling efficiency of nearly 100%. Limited or no uptake was observed for cells incubated with ferumoxides or MION-46L alone. For TA-SPIO-labeled cells, MR images and relaxometry findings showed a 50%-90% decrease in signal intensity and a more than 40-fold increase in T2s. Cell viability varied from 103.7% +/- 9 to 123.0% +/- 9 compared with control cell viability at 9 days, and cell proliferation was not affected by endosomal incorporation of SPIO nanoparticles. Iron concentrations varied with ferumoxides-TA combinations and cells with a maximum of 30.1 pg +/- 3.7 of iron per cell for labeled mesenchymal stem cells. CONCLUSION: Magnetic labeling of mammalian cells with use of ferumoxides and TAs is possible and may enable cellular MR imaging and tracking in experimental and clinical settings.     

Magnetic nanoparticles for imaging dendritic cells

We report the development of superparamagnetic iron oxide (SPIOs) nanoparticles and investigate the migration of SPIO‐labeled dendritic cells (DCs) in a syngeneic mouse model using magnetic resonance (MR) imaging. The size of the dextran‐coated SPIO is roughly 30 nm, and the DCs are capable of independent uptake of these particles, although not at levels comparable to particle uptake in the presence of a transfecting reagent. On average, with the assistance of polylysine, the particles were efficiently delivered inside DCs within one hour of incubation. The SPIO particles occupy approximately 0.35% of cell surface and are equivalent to 34.6 pg of iron per cell. In vivo imaging demonstrated that the labeled cells migrated from the injection site in the footpad to the corresponding popliteal lymph node. The homing of labeled cells in the lymph nodes resulted in a signal drop of up to 79%. Furthermore, labeling DCs with SPIO particles did not compromise cell function, we demonstrated that SPIO‐enhanced MR imaging can be used to track the migration of DCs effectively in vivo. Magn Reson Med 63:1383–1390, 2010. © 2010 Wiley‐Liss, Inc.     

Capacity of human monocytes to phagocytose approved iron oxide MR contrast agents in vitro

To evaluate the capacity of human monocytes to phagocytose various approved iron oxide based magnetic resonance (MR) contrast agents and to optimize in vitro labeling of these cells. Human monocytes were incubated with two superparamagnetic iron oxide particles (SPIO) as well as two ultrasmall SPIO (USPIO) at varying iron oxide concentrations and incubation times. Iron uptake in monocytes was proven by histology, quantified by atomic emission absorption spectrometry and depicted with T2* weighted fast field echo (FFE) MR images at 1.5 T. Additionally, induction of apoptosis in iron oxide labeled monocytes was determined by YO-PRO-1 staining. Cellular iron uptake was significantly (P<0.01) higher after incubation with SPIO compared with USPIO. For SPIO, the iron oxide uptake was significantly (P<0.01) higher after incubation with the ionic Ferucarbotran as compared with the non-ionic Ferumoxides. Efficient cell labeling was achieved after incubation with Ferucarbotran at concentrations 500 g Fe/ml and incubation times 1 h, resulting in a maximal iron oxide uptake of up to 50 pg Fe/cell without impairment of cell viability. In vitro labeling of human monocytes for MR imaging is most effectively obtained with the approved SPIO Ferucarbotran. Potential subsequent in vivo cell tracking applications comprise, e.g. specific targeting of inflammatory processes.     

Characterization of biophysical and metabolic properties of cells labeled with superparamagnetic iron oxide nanoparticles and transfection agent for cellular MR imaging

PURPOSE: To evaluate the effect of using the ferumoxides-poly-l-lysine (PLL) complex for magnetic cell labeling on the long-term viability, function, metabolism, and iron utilization of mammalian cells. MATERIALS AND METHODS: PLL was incubated with ferumoxides for 60 minutes, incompletely coating the superparamagnetic iron oxide (SPIO) through electrostatic interactions. Cells were coincubated overnight with the ferumoxides-PLL complex, and iron uptake, cell viability, apoptosis indexes, and reactive oxygen species formation were evaluated. The disappearance or the life span of the detectable iron nanoparticles in cells was also evaluated. The iron concentrations in the media also were assessed at different time points. Data were expressed as the mean +/- 1 SD, and one-way analysis of variance and the unpaired Student t test were used to test for significant differences. RESULTS: Intracytoplasmic nanoparticles were stained with Prussian blue when the ferumoxides-PLL complex had magnetically labeled the human mesenchymal stem and HeLa cells. The long-term viability, growth rate, and apoptotic indexes of the labeled cells were unaffected by the endosomal incorporation of SPIO, as compared with these characteristics of the nonlabeled cells. In nondividing human mesenchymal stem cells, endosomal iron nanoparticles could be detected after 7 weeks; however, in rapidly dividing cells, intracellular iron had disappeared by five to eight divisions. A nonsignificant transient increase in reactive oxygen species production was seen in the human mesenchymal stem and HeLa cell lines. Labeled human mesenchymal stem cells did not differentiate to other lineage. A significant increase in iron concentration was observed in both the human mesenchymal stem and HeLa cell media at day 7. CONCLUSION: Magnetic cellular labeling with the ferumoxides-PLL complex had no short- or long-term toxic effects on tumor or stem cells.     

Cell motility of neural stem cells is reduced after SPIO‐labeling,which is mitigated after exocytosis

MRI is used for tracking of superparamagnetic iron oxide (SPIO)‐labeled neural stem cells. Studies have shown that long‐term MR tracking of rapidly dividing cells underestimates their migration distance. Time‐lapse microscopy of random cellular motility and cell division was performed to evaluate the effects of SPIO‐labeling on neural stem cell migration. Labeled cells divided symmetrically and exhibited no changes in cell viability, proliferation, or apoptosis. However, SPIO‐labeling resulted in decreased motility of neural stem cells as compared with unlabeled controls. When SPIO‐labeled neural stem cells and human induced pluripotent stem cells were transplanted into mouse brain, rapid exocytosis of SPIO by live cells was observed as early as 48 h postengraftment, with SPIO‐depleted cells showing the farthest migration distance. As label dilution is negligible at this early time point, we conclude that MRI underestimation of cell migration can also occur as a result of reduced cell motility, which appears to be mitigated following SPIO exocytosis. Magn Reson Med, 2013. © 2012 Wiley Periodicals, Inc.     

MR tracking of transplanted cells with "positive contrast" using manganese oxide nanoparticles

Rat glioma cells were labeled using electroporation with either manganese oxide (MnO) or superparamagnetic iron oxide (SPIO) nanoparticles. The viability and proliferation of SPIO-labeled cells (1.9 mg Fe/ml) or cells electroporated with a low dose of MnO (100 microg Mn/ml) was not significantly different from unlabeled cells; a higher MnO dose (785 microg Mn/ml) was found to be toxic. The cellular ion content was 0.1-0.3 pg Mn/cell and 4.4 pg Fe/cell, respectively, with cellular relaxivities of 2.5-4.8 s(-1) (R(1)) and 45-84 s(-1) (R(2)) for MnO-labeled cells. Labeled cells (SPIO and low-dose MnO) were each transplanted in contralateral brain hemispheres of rats and imaged in vivo at 9.4T. While SPIO-labeled cells produced a strong "negative contrast" due to the increase in R(2), MnO-labeled cells produced "positive contrast" with an increased R(1). Simultaneous imaging of both transplants with opposite contrast offers a method for MR "double labeling" of different cell populations.     

In vivo MR imaging of intravascularly injected magnetically labeled mesenchymal stem cells in rat kidney and liver

PURPOSE: To evaluate in vivo magnetic resonance (MR) imaging with a conventional 1.5-T system for depiction and tracking of intravascularly injected superparamagnetic iron oxide (SPIO)-labeled mesenchymal stem cells (MSCs). MATERIALS AND METHODS: This study was conducted in accordance with French law governing animal research and met guidelines for animal care and use. Rat MSCs were labeled with SPIO and transfection agent. Relaxation rates at 1.5 T, cell viability, proliferation, differentiation capacity, and labeling stability were assessed in vitro as a function of SPIO concentration. MSCs were injected into renal arteries of healthy rats (labeled cells in four, unlabeled cells in two) and portal veins of rats treated with carbon tetrachloride to induce centrolobular liver necrosis (labeled cells and unlabeled cells in two each). Follow-up serial T2*-weighted gradient-echo MR imaging and R2* mapping were performed. MR imaging findings were compared histologically. RESULTS: SPIO labeling caused a strong R2* effect that increased linearly with iron dose; R2* increase for cells labeled for 48 hours with 50 microg of iron per milliliter was 50 sec(-1) per million cells per milliliter. R2* was proportional to iron load of cells. SPIO labeling did not affect cell viability (P > .27). Labeled cells were able to differentiate into adipocytes and osteocytes. Proliferation was substantially limited for MSCs labeled with 100 microg Fe/mL or greater. Label half-life was longer than 11 days. In normal kidneys, labeled MSCs caused signal intensity loss in renal cortex. After labeled MSC injection, diseased liver had diffuse granular appearance. Cells were detected for up to 7 days in kidney and 12 days in liver. Signal intensity loss and fading over time were confirmed with serial R2* mapping. At histologic analysis, signal intensity loss correlated with iron-loaded cells, primarily in renal glomeruli and hepatic sinusoids; immunohistochemical analysis results confirmed these cells were MSCs. CONCLUSION: MR imaging can aid in monitoring of intravascularly administered SPIO-labeled MSCs in vivo in kidney and liver.     

低浓度超顺磁性氧化铁标记兔骨髓间充质干细胞的体外磁共振成像研究

目的:以浓度为25μg Fe/ml的超顺磁性氧化铁纳米粒子(SPIO)体外标记兔骨髓间充质干细胞(BMSCs),并探讨1.5 T核磁共振仪成像的特征和成像所需最低标记细胞浓度,以及在标记后1 d、1周、2周、3周、4周的信号变化特征。方法:分离、纯化、培养兔BMSCs并以25μg Fe/ml的SPIO培养液浓度标记,对标记后不同时间的细胞行普鲁士蓝染色和台盼蓝拒染后显微镜观察,并进行MR成像,测量不同序列下不同浓度标记细胞管的信号强度,以确定扫描敏感序列及成像所需最低标记细胞浓度;再测量不同细胞浓度不同时相信号强度,来观察信号强度随时间变化的规律,并进行统计学分析。结果:浓度为25μg Fe/ml的超顺磁性氧化铁纳米粒子标记BMSCs的有效率接近100%,普鲁士蓝染色见细胞浆内有大小不等的蓝染铁颗粒,且在标记后4周内细胞仍具有活力,标记后的BMSCs在T2WI、尤其是GRE(T2*WI)序列信号明显降低;并且细胞浓度越高信号降低越明显,GRE序列MR成像的最低细胞浓度为5×104/ml。当标记细胞浓度为5×104/ml时,信号在T2*WI序列的降低2周后失去统计学意义;而在细胞浓度为5×105/ml时,标记3周后,信号在T2*WI序列的降低才失去统计学意义。结论:25μg/ml铁浓度标记干细胞不仅标记效率高,而且对细胞生长及增殖活力无明显影响,标记后MR信号改变与干细胞数目及标记时间相关。