摘要
目的:
合成一种能用于分子影像学的新型纳米造影剂Gd(OH)_3，并对其样本进行表征。初步研究Gd(OH)_3作为磁共振造影剂的毒性，并探讨其弛豫效能、在健康小鼠体内的分布情况及对荷前列腺癌鼠瘤体显示情况。为进一步研究其长期毒性及评估其临床应用的可行性提供依据。
材料与方法：
（1）将Gd(NO_3)_3·6H_2O（10mmol）溶于20mL去离子水中，搅拌后得到均匀的溶液。在不断搅拌的条件下，通过连续滴定将2.5mol/L的NaOH溶液滴入上述溶液中，补加去离子水使得溶液的体积达到45mL，然后将此溶液倒入一个体积为50mL有Teflon（特氟龙）内衬的不锈钢高压釜内，将上盖拧紧，放入烘箱中升温至180℃，在该温度下反应24小时，然后将高压釜自然冷却至室温。得到的产物为白色粉末，用纱布包好后放入索氏提取器中，先用去离子水洗3-4遍，再用乙醇清洗3-4遍后，在60℃恒温条件下真空烘箱中抽真空24小时，以备后续实验研究用。应用扫描电镜(SEM)、透射电镜(TEM)、X-射线衍射(XRD)分析对样本进行表征。（2）应用MTT法行体外细胞毒性实验，将HepG2和MCF-7细胞制成单细胞悬液,接种5×10~3个细胞/孔于96孔细胞培养板上,37℃，5%CO_2的培养箱内培养12h。加入含10%血清的培养基配制的纳米粒子悬浮液，终浓度分别为200μg/mL、100μg/mL、50μg/mL、25μg/mL、12.5μg/mL、6.25μg/mL、3.125μg/mL、0μg/mL，每组6复孔。同等条件下将两种细胞分别培养48h。加MTT（5mg/mL）溶液20μL，继续培养4h。小心吸弃上清，每孔加入150μL DMSO（二甲基亚砜），震荡摇匀40min，在570nm下测定吸光度值(应用Bio-Rad model-68酶标仪)，计算细胞生存率，分析细胞活力。为了进行体外细胞形态分析，将密度2×10_4的HepG2细胞放在12孔平板上4小时让细胞充分粘附。将所有的细胞用冷的磷酸盐缓冲溶液(PBS)冲洗之后，将纳米粒子(200μg/mL)加入到细胞培养基中。在培养48h之后，再将细胞用磷酸盐缓冲溶液冲洗几次以除去剩余的纳米粒子，然后使用光学显微镜（奥林巴斯BX-51）在白光下观察。体内动物实验设实验组和对照组，每组各6只小白鼠，分别注射Gd(OH)_3和0.9%NaCL。为进行组织学分析，两组小鼠饲养30天后被解剖，将器官(心脏、脾、肝、肺及肾)按照对照组和实验组分组，并且浸泡在10%中性福尔马林中，再将收集起来的器官封藏在石蜡中，切片4μm厚，使用H&E染料染色，在光学显微镜下观察组织切片。体外T_1WI磁共振成像使用1.5T临床磁共振成像仪，将不同浓度的Gd(OH)_3纳米粒子稀释液加入到包含1%琼脂糖的磷酸盐缓冲溶液中，作为对比剂放在一系列4.0mL埃彭道夫管中用于T_1WI磁共振成像。（3）12只小鼠随机分成2组，每组各6只。对照组注射Gd-DTPA，实验组注射Gd(OH)_3,两组均行1.5T磁共振T_1WI、T_2WI平扫及T_1WI增强扫描。增强扫描时间点选择在注射对比剂后15min、30min、1h、2h、4h、6h、8h、16h和24h。测量肾脏皮质、肝脏平扫及增强各个时间点T_1WI信号强度。（4）建立前列腺癌小鼠皮下移植瘤动物模型，12只荷前列腺癌小鼠随机分为2组，每组6只，分别注射Gd-DTPA和Gd(OH)_3。两组均行1.5T磁共振T_1WI、T_2WI平扫及T_1WI增强扫描。增强扫描时间点选择在注射对比剂后15min、30min、1h、2h、4h、6h、8h、16h和24h。测量2组实验动物肿瘤实质部分平扫和增强后各个时间点的T_1WI信号强度。测量背景噪声，计算2组平扫及增强各个时间点肿瘤组织的信噪比。
结果：
（1）我们成功制备了Gd(OH)_3纳米粒子并对其进行了形貌表征。从Gd(OH)_3纳米粒子的扫描电子显微镜(SEM)图像中，可以清楚看到样品纳米粒子表面光滑，单个分散，长度大致相同，尺寸在100nm左右，直径均一，尺寸在15nm左右。（2）MTT实验表明在有纳米粒子的条件下培养48小时，仍然有90%以上的细胞能存活。实验组和对照组所有鼠在饲养30天后处死。没有观察到与Gd(OH)_3纳米粒子的处理有关的组织损伤以及任何其他不利影响。随着外加磁场强度的增加，纳米粒子的磁化强度与外加磁场强度呈理想线性关系，说明Gd(OH)_3纳米粒子拥有顺磁性。制备好的纳米粒子测得的最大磁化率值为8.9×10~(-5)emu/g~(-1)Oe~(-1)。从弛豫率（1/T_1）随Gd3+浓度变化图斜率计算出的纳米粒子的相对r1值为12.3mM~(-1)S~(-1)（。3）两组肝脏和肾脏平扫T_1WI信号强度均无统计学差异。两组肝脏在注射造影剂后15min均有强化，信号均匀，此后Gd-DTPA组肝脏信号强度逐渐下降，Gd(OH)_3组肝脏信号强度持续至1h后逐渐下降。注射造影剂后两组间相同时间点肝脏T_1WI信号值差异有统计学意义，同组不同时间点之间T_1WI信号值有差异(F=20944.4，p<0.0001)，二者有交互效应(F=2359.96，p<0.0001)，说明随着时间的变化，两组各时间点信号值变化趋势不同,随时间延长，Gd(OH)_3组信号值降低幅度小于Gd-DTPA组。两组肾脏增强后15min均显示皮质均匀强化。15min后Gd-DTPA组信号明显下降，Gd(OH)_3组信号强度也有下降，但不如Gd-DTPA组明显，并且8h到16h出现一个显著平台期，造影剂廓清速度明显减慢。增强后两组间相同时间点肾脏T_1WI信号值差异有统计学意义，同组不同时间点之间肾脏T_1WI信号值有差异(F=3001.83，p<0.0001)，二者有交互效应(F=1279.4，p<0.0001)，说明随着时间的变化，两组各时间点信号值变化趋势不同。随时间延长，Gd(OH)_3组信号值降低幅度小于Gd-DTPA组。(4)前列腺癌皮下移植瘤模型建立成功。瘤体在MRI平扫均被检查出。均呈类圆形，直径约为15mm至20mm，平扫T_1WI均为低信号，T_2WI均为高信号，T_1WI和T_2WI信号均较均匀，两组肿瘤在增强后15min均有强化，Gd-DTPA组肿瘤在注射造影剂15min后T_1WI信号强度逐渐下降，至24h仍略高于平扫T_1WI信号值，Gd(OH)_3组信号强度在注射造影剂15min后逐渐上升，至6h达到峰值，此后信号逐渐下降，至24h仍略高于平扫T_1WI信号值。肿瘤平扫时两组T_1WI信号值差异无统计学意义。两组间注射造影剂后相同时间点肿瘤T_1WI信号值差异有统计学意义，同组不同时间点之间肿瘤T_1WI信号值有差异(F=27734.9，p<0.0001)，二者有交互效应(F=4630.27，p<0.0001)，说明随着时间的变化，两组各时间点信号值变化趋势不同。随时间延长，Gd(OH)_3组信号值降低幅度小于Gd-DTPA组。两组间平扫及注射造影剂15min后肿瘤信号噪声比差异无统计学意义，注射造影剂后其余各个时间点信号噪声比差异有统计学意义。同组不同时间点之间肿瘤信号噪声比有差异(F=377.39，p<0.0001)，二者有交互效应(F=168.82，p<0.0001)，说明随着时间的变化，两组各时间点肿瘤信号噪声比变化趋势不同。随时间延长，Gd(OH)_3组肿瘤信号噪声比降低幅度小于Gd-DTPA组。
结论：
我们用一种简单易行的、产出率高的水热法合成了纳米磁共振造影剂Gd(OH)_3，对其进行了详细表征分析，评价了该纳米粒子的特性。细胞毒性实验和注射该纳米粒子后的组织学分析进一步表明其有很好的组织相容性，表明其有用于疾病诊断的可能性。体外MR成像研究证实其比商用Gd-DTPA复合物有更高的弛豫率。Gd(OH)_3在正常小鼠体内成像及荷前列腺癌鼠肿瘤成像的实验研究，初步明确了其在正常鼠体内的分布和代谢情况及其与传统造影剂比较对肿瘤显影有明显的优势。实验表明纳米磁共振造影剂Gd(OH)_3有广阔的开发前景，其作为临床应用造影剂有巨大的潜力，对恶性肿瘤诊断存在潜在的巨大价值。因为其作为纳米造影剂的易修饰特点，如果加载特异性配体会实现对肿瘤的特异性诊断，我们正在进行这方面深入地研究。
Objective:
To synthesize a novel gadolinum-loaded nanorods as a molecular imagingcontrast agent for magnetic resonance imaging.And to study the toxiciy, relax-ativity, ditribution in normal mice,MR imaging of carcinoma of prostate inmice with this novel cntrast media.
Material and Methods:
(1) The monodispersed Gd(OH)_3nanorods were fabricated by one-pothydrothermal method. Typically, Gd(NO_3)_3·6H_2O (10mmol) was dissolvedinto deionized water (20mL) to form a homogeneous solution. A NaOHaqueous solution (2.5M) was added into above solution under vigorouslymagnetic stirring through continuous titration until the pH value reached12.The final volume was then adjusted to45mL by adding deionized water andthe resulting solution was sealed to a Teflon-lined stainless-steel autoclave (50mL). After reaction for24h at180℃, the autoclave was cooled to ambienttemperature naturally.（2）MTT reduction assays were carried out to quantifiedthe cytotoxicity of Gd(OH)_3nanorods. In a typical procedure, cells (HepG2andMCF-7cell lines) were cultured in96-well plates as a density of5000per wellfor12h to allow the cells to attach. Subsequently, serial dilutions of differentnanorod formulations were added to the culture medium. At the end of theincubation time, the medium containing nanorods were removed, and cellsamples were treated with MTT for another4h, which was followed by theaddition of dimethyl sulfoxide (DMSO) to dissolve the formazan crystals.Bio-Rad model-680microplate reader was applied to measure the absorbanceat a wavelength of570nm (corrected for background absorbance at490nm). Six replicates were done for each treatment group and percent viability wasnormalized to cell viability in the absence of nanorods.In a typical experiment,HepG2cells with a density of2×10_4were plated in a12-well plate for4h toallow the cells to attach. After the cells were washed twice by coolphosphate-buffered saline (PBS), nanorods (200μg/mL) were added to the cellculture medium. After incubating for48h, the cells were washed again withPBS several times to remove the remaining nanorods, and then observed underan Olympus BX-51optical system microscopy.Mice were sacrificed30daysafter administration for the histology studies. The tissues (heart, spleen, liver,lung, and kidney)were collected from two groups (contro and test group).Invitro T_1-weighted MR imaging was acquired by using a1.5T clinical MRIinstrument. Dilutions of Gd(OH)_3nanorods in PBS buffer containing1%agarose with expected different concentrations as contrast agent were placed ina series of4.0mL Eppendorf tubes for T_1-weighted MR imaging and fixed in10%neutral buffered formalin. For in vivo MR imaging, Gd(OH)_3nanorodswere dispersed in0.9wt%NaCL solution, then injected into the mice throughthe tail vein.(3)12mice were randomly divided into2groups and administeredwith two contrast agents: Gd-DTPA and Gd(OH)_3.1.5Tesla MR seanner wereused. The Plain T_1WI SE sequences were performed.The repeated T_1WI SEsequences were then Performed15min,30min, l h,2h,4h,6h,8h,12h,18h and24h after the intravenous injection of Gd-DTPA and Gd(OH)_3. Thesignal intensity of the liver,and renal cortex in the pre-and post-contrast T_1WIwas measured using a region of interesting with2mm_2.（4）The prostatecarcinoma cell were inoculated subeutaneously in mice to produce prostatecarcinoma model.12mice were randomly divided into two groups andadministered with Gd-DTPA and Gd(OH)_3. MR T_2WI FSE sequence and T_1WISE sequences before and after the intravenous injection of two contrast agentswere performed.The scan parameters and the time points were the same as last part. The T_1WI signal intensity of the tumor was measured and the value ofsignal to noise ratio (SNR) was calculated.
Results:
(1) For the fabrication of Gd(OH)_3nanorods, a vigorous hydrothermalmethod has been introduced to perform the experiment. Scanning electronmicroscopy (SEM) image for the pure Gd(OH)_3nanorods confirm that thesample consists of monodisperse rod-like nanoparticles with a mean length of100nm, an average diameter of15nm, as well as smooth surface.(2) MTTassay showde the cell viability results, revealing that more than90%cells weresurvived even after48h of incubation with the nanorods. The mice of the testand control groups were sacrificed30days after administration. No tissuedamage or any other adverse effect associated with the administration of theGd(OH)_3nanorods were observed. The mass magnetic susceptibility value ofthe as-prepared nanorods was determined to be8.9×10~(-5)emu/g Oe. The T_1WIimages were evaluated at a1.5T human clinical scanner and the relaxivity r1value of the nanorods calculated from the slope of the plot of the relaxation rate(1/T_1) as a function of Gd3+concentration was determined to be12.3mM~(-1)S~(-1).Along with the strength of the applied magnetic field increased, ideal linearcorrelation between the magnetization and the applied magnetic field in thenanorods was obtained, demonstrating that Gd(OH)_3nanorods possessedparamagnetism.(3) The signal value of T_1WI of the liver and kidney was notstastistically different when no enhacement. The signal value of T_1WI of theliver was stastistically different at the time point of15min,30min, l h,2h,4h,6h,8h,12h,18h and24h among two groups. The signal value of T_1WI of thekidney was not stastistically different when no enhacement. The signal value ofT_1WI of the kidney was stastistically different at the time Point of15min,30min,l h,2h,4h,6h,8h,12h,18h and24h among two groups.(4) Themodel of the prostate carcinoma was successfully obtained in12mice. All the prostate carcinoma were detected by MR and the range of the tumor size wasfrom15mm to20mm. The tumor is low signal on T_1WI and high signal onT_2WI. The signal value of T_1WI of the tumor was not stastistically differentwhen no enhacement. The signal value of T_1WI of the tumor was stastisticallydifferent at the time Point of15min，30min，l h,2h,4h,6h,8h,12h,18h and24h among two groups. The SNR of the tumor was not stastistically differentwhen no enhacement and15min.The SNR of the tumor was stastisticallydifferent at the time point of30min,l h,2h,4h,6h,8h,12h,18h and24hamong two groups.
Conclusions:
In summary, we have synthesized monodisperse Gd(OH)_3nanorodsthrough a facile and scalable hydrothermal route. Detailed characterizationshave been performed to evaluate the properties of the nanorods. Cell-cytotoxicity assay and post-injection histology analysis further demonstrate theexcellent biocompatibility of the nanorods, indicating the feasibility for diseasediagnosis and chemotherapy. MRI measurement studies of the Gd(OH)_3nanorods revealed a higher T_1-weight relaxation rate compared withcommercial Gd-DTPA complex. In addition, the capability of nanorods ascontrast agents for MR imaging has been evaluated through preliminary in vivocharacterization. The advantage of the Gd(OH)_3for the malignant prostatetumor of mice is very apparent. Due to these advantages, the well-constructedGd(OH)_3nanorods are promising for MR imaging, showing more potentials forfurther clinical application. The specific functionalization of the as-preparednanorods for specific targeting is in progress, and research extended for moredetailed in vivo live animal systems are also undergoing.
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