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.     

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.     

胎鼠神经干细胞超顺磁性氧化铁颗粒标记移植后MRI研究

目的:探讨超顺磁性氧化铁颗粒(SPIO)标记神经干细胞的方法,以及标记细胞正常大鼠脑内移植后MR成像的方法学研究。方法:多聚左旋赖氨酸介导的SPIO标记胎鼠神经干细胞,进行台盼兰染色和普鲁士兰染色分别检测标记细胞的存活率和标记率。选取SD大鼠15只,简单随机法分为3组:第1组于大鼠右侧尾状核移植未标记的NSCs,第2组于大鼠右侧尾状核移植标记的NSCs,第3组右侧尾状核移植游离的SPIO颗粒,移植后第1、4、8周进行MRI。8周后处死大鼠,行组织切片普鲁士兰染色。结果:体外标记的神经干细胞普鲁士兰染色发现铁颗粒聚集于细胞浆内,标记率为100%;标记细胞与未标记细胞的台盼兰染色结果无显著差异。移植后MRI,第1组注射点未见低信号影;第2组注射点T2WI及GRE序列均可见类圆形低信号影;第3组大鼠注射后1周注射点可见低信号影,4周后低信号影变淡且边缘变模糊,8周后低信号影T2WI已不明显。与T2WI序列比较,GRE序列显示标记细胞更清晰,但显示范围较扩散。脑组织切片的普鲁士兰染色显示,第1组大鼠脑组织切片未见异常蓝染细胞,第2组注射点可见蓝染细胞,第3组注射点可见稍许散在蓝色颗粒状物质。结论:多聚左旋赖氨酸介导下SPIO可用于标记神经干细胞,标记细胞移植后MRI可以无创性观察移植神经干细胞的位置及分布情况。     

Comparison of SPIO and USPIO for in vitro labeling of human monocytes: MR detection and cell function

PURPOSE: To label human monocytes with superparamagnetic iron oxide (SPIO) and compare labeling efficiency with that of ultrasmall SPIO (USPIO) and evaluate the effect of iron incorporation on cell viability, migratory capacity, and proinflammatory cytokine production. MATERIALS AND METHODS: The study was approved by the institutional ethics committee; informed consent was obtained from donors. Freshly isolated human monocytes were labeled with iron particles of two sizes, USPIOs of 30 nm and SPIOs of 150 nm, for 1.5 hours in culture medium containing 0.1, 0.5, 1.0, and 3.7 mg of iron per milliliter. Labeling efficiency was determined with relaxation time magnetic resonance (MR) imaging (4.7 T) and Prussian blue staining for presence of intracellular iron. Cell viability was monitored; migratory capacity of monocytes after labeling was evaluated by using an in vitro assay with monolayers of brain endothelial cells. Levels of proinflammatory cytokines, interleukin (IL) 1 and IL-6, were measured with enzyme-linked immunosorbent assay 24 hours after labeling. Data were analyzed with Student t test or two-way analysis of variance followed by a multiple-comparison procedure. RESULTS: R2 relaxation rates increased for cell samples incubated with SPIOs, whereas rates were not affected for samples incubated with highest concentration of USPIOs. Labeling monocytes with SPIOs (1.0 mg Fe/mL) resulted in an R2 of 13.1 sec(-1) +/- 0.8 (standard error of the mean) (7 sec(-1) +/- 0.2 for vehicle-treated cells, P < .05) and had no effect on cell viability. On the basis of T2 relaxation times, the in vitro MR detection limit of 58 labeled monocytes per 0.05 microL was calculated. Migration of labeled monocytes was not different from that of vehicle-treated cells. Intracellular iron had no effect on production of IL-1 and IL-6 24 hours after labeling. CONCLUSION: In vitro labeling of human monocytes is effective by using SPIOs, not USPIOs. Incubation with SPIOs (1.0 mg Fe/mL) results in efficient labeling detectable on MR images and does not affect cellular viability and activation markers such as cell migration and cytokine production.     

Labeling efficacy of superparamagnetic iron oxide nanoparticles to human neural stem cells: comparison of ferumoxides, monocrystalline iron oxide, cross-linked iron oxide (CLIO)-NH2 and tat-CLIO.

OBJECTIVE: We wanted to compare the human neural stem cell (hNSC) labeling efficacy of different superparamagnetic iron oxide nanoparticles (SPIONs), namely, ferumoxides, monocrystalline iron oxide (MION), cross-linked iron oxide (CLIO)-NH(2) and tat-CLIO. MATERIALS AND METHODS: The hNSCs (5 x 10(5) HB1F3 cells/ml) were incubated for 24 hr in cell culture media that contained 25 microg/ml of ferumoxides, MION or CLIO-NH(2), and with or without poly-L-lysine (PLL) and tat-CLIO. The cellular iron uptake was analyzed qualitatively with using a light microscope and this was quantified via atomic absorption spectrophotometry. The visibility of the labeled cells was assessed with MR imaging. RESULTS: The incorporation of SPIONs into the hNSCs did not affect the cellular proliferations and viabilities. The hNSCs labeled with tat-CLIO showed the longest retention, up to 72 hr, and they contained 2.15+/-0.3 pg iron/cell, which are 59 fold, 430 fold and six fold more incorporated iron than that of the hNSCs labeled with ferumoxides, MION or CLIO-NH(2), respectively. However, when PLL was added, the incorporation of ferumoxides, MION or CLIO-NH(2) into the hNSCs was comparable to that of tat-CLIO. CONCLUSION: For MR imaging, hNSCs can be efficiently labeled with tat-CLIO alone or with a combination of ferumoxides, MION, CLIO-NH(2) and the transfection agent PLL.     

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.     

大鼠骨髓间充质干细胞磁标记及MR成像研究

目的应用菲立磁．多聚左旋赖氨酸复合物标记大鼠骨髓间充质干细胞，探讨MR成像显示磁标记干细胞的可行性。方法制备菲立磁-多聚左旋赖氨酸复合物。分离培养Wistar大鼠骨髓间充质干细胞，以菲立磁-多聚左旋赖氨酸复合物标记干细胞。分别于标记后24h及1、2、3周行普鲁士蓝染色观察细胞内铁，台盼蓝排除试验检测细胞活力。应用1．5TMR仪，以SE序列T1WI、T2WI和梯度回波（GRE）序列T2＊WI行磁标记干细胞成像。结果普鲁士蓝染色显示细胞质内大量铁颗粒存在，标记率100％；随细胞分裂增殖，细胞内铁颗粒逐渐减少。干细胞磁标记后24h及1、2、3周的台盼蓝拒染率分别为91．00％、93．00％、91．75％和92．50％，与未标记细胞相比较差异无统计学意义（P〉0．05）。10^3、10^4、10^5个磁标记干细胞T2WI信号降低分别为63．75％、82．31％、91．92％，T2＊WI信号降低分别为68．24％、83．01％、93．94％。10^5个干细胞磁标记后24h及1、2、3周T2＊WI信号降低分别为93．75％、75．92％、41．75％、8．83％。结论应用菲立磁-多聚左旋赖氨酸复合物标记大鼠间充质干细胞安全、有效；T2＊WI对磁标记干细胞的显示最敏感；MR信号改变与干细胞数目及分裂增殖状态相关。     

骨髓间充质干细胞的培养和超顺磁氧化铁标记

目的探讨全血贴壁法培养成年大鼠骨髓间充质于细胞的可行性，寻找一种对间充质干细胞进行示踪的方法。方法取6～8周龄雄性SD大鼠胫骨、股骨，收集骨髓腔冲洗悬液直接培养，经多次换液、传代，免疫组化法检测CD34、CD44表达情况。以超顺磁氧化铁和P1代间充质干细胞共同孵育培养24h，普鲁士蓝染色评价标记效率，台盼蓝染色检验标记后细胞的活性。结果全血贴壁法培养的大鼠间充质干细胞贴壁，呈纤维细胞样生长，CD44阳性细胞比率为96％，不表达CD34。经超顺磁氧化铁标记后，普鲁士蓝染色标记效率为90％，标记后98％的细胞保持活性。结论全血贴壁法能成功培养大鼠间充质干细胞，超顺磁氧化铁可以安全、有效地标记间充质干细胞。     

Combination of transfection agents and magnetic resonance contrast agents for cellular imaging: relationship between relaxivities, electrostatic forces, and chemical composition.

The purpose of this study was to investigate the changes in electrostatic and magnetic resonance (MR) properties observed when MR contrast agents (CAs) (Feridex, MION-46L, or G5-dendrimer-DOTA-Gd) are combined with transfection agents (TAs) under various conditions for use as a CA-TA complex basis for cellular labeling and MRI. CAs were incubated with various classes of TAs for 0-48 hr in solutions of varying concentrations and pH values. NMR relaxation rates (1/T(1), 1/T(2)), MRI and zeta potential (ZP) of CA-TA solutions were measured. TAs decreased the 1/T(1) and 1/T(2) of G5-DOTA-Gd, Feridex, and MION-46L by 0-95%. Altering the pH of G5-DOTA-Gd-TA decreased the T(1)-weighted signal intensity (SI) on MRI from 0 to 78%. Measured ZP values for G5-DOTA-Gd, Feridex, and MION-46L were -51, -41, and -2.0 mV, respectively. The TA LV had a negative ZP, while the other TAs had ZPs ranging from +20 to +65 mV. The alteration of the ZP and NMR relaxivities of the MR CAs, Feridex, MION-46L, and G5-DOTA-Gd by TAs has been demonstrated. These results enhance our understanding of the relationship between electrostatic and MR properties.     

R2 and R2* mapping for sensing cell-bound superparamagnetic nanoparticles: in vitro and murine in vivo testing

PURPOSE: To prospectively determine the cellular iron uptake by using R2 and R2* mapping with multiecho readout gradient-echo and spin-echo sequences. MATERIALS AND METHODS: All experiments were approved by the institutional animal care committee. Lung carcinoma cells were lipofected with superparamagnetic iron oxides (SPIOs). Agarose gel phantoms containing (a) 1 x 10(5) CCL-185 cells per milliliter of agarose gel with increasing SPIO load (0.01-5.00 mg of iron per milliliter in the medium), (b) different amounts (5.0 x 10(3) to 2.5 x 10(5) cells per milliliter of agarose gel) of identically loaded cells, and (c) free (non-cell-bound) SPIOs at the iron concentrations described for (b) were analyzed with 3.0-T R2 and R2* relaxometry. Iron uptake was analyzed with light microscopy, quantified with atomic emission spectroscopy (AES), and compared with MR data. For in vivo relaxometry, agarose gel pellets containing SPIO-labeled cells, free SPIO, unlabeled control cells, and pure agarose gel were injected into three nude mice each. Linear and nonlinear regression analyses were performed. RESULTS: Light microscopy and AES revealed efficient SPIO particle uptake (mean uptake: 0.22 pg of iron per cell +/- 0.1 [standard deviation] for unlabeled cells, 31.17 pg of iron per cell +/- 4.63 for cells incubated with 0.5 mg/mL iron). R2 and R2* values were linearly correlated with cellular iron load, number of iron-loaded cells, and content of freely dissolved iron (r(2) range, 0.92-0.99; P < .001). For cell-bound SPIO, R2* effects were significantly greater than R2 effects (P < .01); for free SPIO, R2 and R2* effects were similar. In vivo relaxometry enabled accurate prediction of the number of labeled cells. R2' (R2* - R2) mapping enabled differentiation between cell-bound and free iron in vitro and in vivo. CONCLUSION: Quantitative R2 and R2* mapping enables noninvasive estimations of cellular iron load and number of iron-labeled cells. Cell-bound SPIOs can be differentiated from free SPIOs with R2' imaging.     

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.     

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.     

Magnetic nanoparticle labeling of mesenchymal stem cells without transfection agent: cellular behavior and capability of detection with clinical 1.5 T magnetic resonance at the single cell level.

The purpose of this work was to evaluate the efficacy of labeling human mesenchymal stem cells (hMSCs) by ionic superparamagnetic iron oxide (SPIO) without a transfection agent and verifying its capability to be detected with clinical 1.5 T magnetic resonance (MR) at the single-cell level. Human hMSCs were incubated for 24 h with an ionic SPIO, Ferucarbotran. The labeling efficiency of hMSCs was determined by iron content measurement spectrophotometrically, and the influence of labeling on cell behavior was ascertained by examination of cell viability using the trypan blue exclusion method, cell proliferation analysis using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, mitochondrial membrane potential (MMP) change, differentiation capacity, and reactive oxygen species (ROS) production measured by dichlorofluorescein diacetate (DCFDA) fluorescent probe. Labeled hMSCs were scanned under 1.5 T MRI with three-dimensional (3D) and two-dimensional (2D) T(2)-weighted gradient echo (GRE) pulse sequences. Human hMSC labeling without transfection agent was efficient. The iron content in hMSCs was 23.4 pg Fe/cell. No significant change was found in viability, proliferation, MMP change, ROS production, or differentiation capacity. About 45.2% of the hMSCs could be detected using 1.5 T MRI at the single cell level with 3D GRE and four repetitions.     

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。     

新型超顺磁性氧化铁标记SD大鼠脂肪干细胞的有效性及安全性研究

目的：采用新型超顺磁性氧化铁对 SD 大鼠来源的脂肪干细胞（ADSCs）进行标记,并与既往商用 SPIO 标记效果进行对比,探讨这种新型超顺磁性氧化铁标记的有效性及安全性。方法：分离、纯化、鉴定 SD 大鼠来源的 ADSCs,然后分不同浓度组（0、6、12、25、50和100μg／mL）和时间组（6、12、24和48 h）进行标记,通过普鲁士蓝染色测定铁标记率;在不影响细胞形态的前提下,对达到95％以上铁染色率的孵育浓度、时间进行标记安全性检测,包括活力、增殖力、细胞表面抗原表达;采用透射电子显微镜观察标记细胞的超微结构,采用 ICP-AES 对标记细胞内的铁含量进行测定,并与商用SPIO 标记效果进行对比。结果：在无细胞毒性的前提下,新型 SPIO 达到95％以上铁染色率的孵育浓度是12和25μg／mL,孵育时间是12 h;ICP-AES 检测显示具有表面正电荷的聚乙二醇（PEG）／聚乙烯亚胺（PEI）修饰的 SPIO 标记后细胞内的铁含量达到35．4 pg／cell（25μg／mL 中孵育12h 后）和20．16 pg／cell（12μg／mL 中孵育12h 后）,并随着孵育浓度的增加,细胞内的铁含量增加;而具有表面零电荷的 PEG／聚乙烯吡咯烷酮（PVP）修饰的 SPIO 标记后的铁含量仅为6．96 pg／cell（25μg／mL 中孵育12h 后）;透射电子显微镜显示标记后细胞器结构完整,内吸收的 SPIO 主要位于细胞质内的囊泡和溶酶体中。结论：新型 SPIO 在适当孵育浓度和时间下可以安全、快速标记 ADSCs;PEG／PEI 修饰的 SPIO 标记效果要远远比既往商用的 SPIO 快速有效,可作为一种优势的新型磁性标记物用于干细胞标记;而 PEG／PVP 修饰的SPIO 比起既往商用的 SPIO 并无明显优势,说明表面电荷在细胞标记中占有极其重要的角色。     

抗血管内皮生长因子MR靶向超顺磁性分子探针的构建及体外大肠癌细胞显像的实验研究

目的 探讨携带纳米铁颗粒的抗血管内皮生长因子(VEGF)分子探针的构建方法及其生物理化性状,以及对体外大肠癌细胞的靶向作用.方法 采用化学交联法构建抗体分子探针,应用十二烷基磺酸钠聚丙烯酰胺凝胶电泳及MR扫描仪检测抗体对比剂的生化及磁学特性.将抗体分子探针与人结肠癌SW620细胞在37℃分别孵育30、60、90 min后进行MR扫描,并进行普鲁士蓝染色验证,信号变化数据组间比较采用单因素方差分析和LSD-t检验;以最佳孵育时间为标准,与骨髓间充质干细胞对照,检测抗体对比剂的靶向增强作用.信号变化数据组间比较采用单因素方差分析和析因设计方差分析.结果 携带纳米铁颗粒的抗VEGF分子探针被成功构建并分离,抗体分子探针的弛豫率为0.0426×10~6 mol/s.免疫细胞荧光及普鲁士蓝染色法证实抗体分子探针可以与高表达VEGF的人结肠癌SW620细胞结合.MRI证明抗体分子探针孵育大肠癌细胞30、60、90 min后T_2信号强度分别为392±7、91±8、264±10,与孵育前(679±12)比较差异有统计学意义(F=4735.489,P<0.01),在孵育60 min后T_2WI及T_2~* WI信号强度值降低最明显.抗体分子探针孵育大肠癌细胞对照实验表明,抗体分子探针孵育大肠癌细胞组与干细胞组T_2信号强度分别为82±7和685±43,差异有统计学意义(t=39.167,P<0.05),抗体分子探针孵育大肠癌细胞组T_2WI及T_2~* WI信号强度显著降低.单纯对比剂孵育大肠癌细胞组与干细胞组比较T_2信号强度分别为419±59和400±41,差异无统计学意义(t=-0.718,P>0.05).结论 携带纳米铁颗粒的抗VEGF分子探针已将肿瘤血管生成的评价发展到受体水平,为肿瘤血管生成的诊断与抗血管生成的治疗提供了新的思路.     

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。     

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.     

In vivo MR tracking of mesenchymal stem cells in rat liver after intrasplenic transplantation

PURPOSE: To prospectively track in vivo in rats intrasplenically transplanted stem cells labeled with superparamagnetic particles by using magnetic resonance (MR) imaging. MATERIALS AND METHODS: The study was approved by the institutional Committee on Animal Research. Liver damage in 12 rats was induced with subcutaneous injection of carbon tetrachloride (CCl4). Intrasplenic transplantation of 6x10(6) rodent bone mesenchymal stem cells (BMSCs) with (n=6) and without (n=6) superparamagnetic particle Fe2O3-poly-L-lysine (PLL) labeling was performed via direct puncture. Cell labeling efficiency was assessed in vitro by using Prussian blue stain and an atomic absorption spectrometer. MR examinations were performed immediately before and 3 hours and 3, 7, and 14 days after transplantation. Liver-to-muscle contrast-to-noise ratios (CNRs) on T2*-weighted MR images obtained before and after injection were measured and correlated with histomorphologic studies. Statistical analyses were performed by using repeated-measures analysis of variance. RESULTS: Rat BMSCs could be effectively labeled with approximately 100% efficiency. Migration of transplanted labeled cells to the liver was successfully documented with in vivo MR imaging. CNRs on T2*-weighted images decreased significantly in the liver 3 hours after injection of BMSCs (P<.05) and returned gradually to the level achieved without labeled cell injection in 14 days. Histologic analyses confirmed the presence of BMSCs in the liver. The labeled cells primarily localized in the sinusoids of periportal areas and the foci of CCl4-induced liver damage. Quantitative analysis of Prussian blue-stained cells indicated gradual decrease of dye pigments from 3 hours to 3, 7, and 14 days after injection. No free iron particles were found in the interstitium or within hepatic microvessels. CONCLUSION: The rat BMSCs could be efficiently labeled with Fe2O3-PLL and the relocation of the labeled cells to rat livers after intrasplenic transplantation could be depicted at in vivo MR imaging.     

肾功能衰竭大鼠超顺磁性氧化铁标记骨髓间充质干细胞肾脏移植MR活体示踪的研究

目的应用磁性氧化铁纳米粒子和多聚左旋赖氨酸（poly-L-lysine，PLL）的偶联物Fe2O3-PLL标记大鼠骨髓间充质干细胞（MSCs），MR活体示踪经肾动脉移植入肾功能衰竭（简称肾衰）大鼠肾脏的标记细胞。方法制备Fe2O3-PLL，分离、纯化并培养大鼠骨髓MSCs，Fe2O3-PLL标记细胞，普鲁士蓝染色显示细胞内铁。肌内注射甘油所致肾衰的大鼠分为2组，分别经左肾动脉移植入标记细胞（6只）和未标记细胞（5只），移植后即刻及第1、3、5、8天应用MRI对移植细胞进行活体示踪，并与肾脏组织切片普鲁士蓝染色和HE染色对照。结果MSCs的Fe2O3-PLL标记率近100％，普鲁士蓝染色显示蓝色铁颗粒位于MSCs胞质内。标记细胞移植后肾衰大鼠肾脏皮质区信号强度明显下降，T2＊WI信号改变最明显，而肾髓质及肾盂信号较细胞移植前无明显变化，信号改变随着时间的延长逐渐减轻一直持续到移植后第8天。组织学分析见绝大多数标记细胞分布于肾皮质肾小球内，与MRI信号改变区域基本一致。未标记细胞移植后未见肾脏信号改变。结论Fe2O3-PLL可以有效标记大鼠骨髓MSCs，临床应用型1．5T磁共振仪可对经肾动脉移植入肾衰大鼠肾脏的标记细胞进行初步活体示踪。