MR网状内皮靶向对比剂Gd-DTPA脂质体的合成及其在肝转移瘤和淋巴成像的应用研究
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摘要
研究目的
1．尝试制备装载Gd-DTPA的脂质体，并对其粒径大小、粒径分布、表面电位进行表征；透射电镜来测定其形态和大小；
2．建立肝转移瘤动物模型，静脉注射Gd-DTPA脂质体，并与游离Gd-DTPA溶液对照，考察网状内皮系统特异性对比剂Gd-DTPA脂质体对提高肝脏转移瘤对比度的价值；
3．建立新西兰白兔腹膜后淋巴结增生模型和子宫内膜癌伴腹膜后淋巴结转移模型，组织间隙注射Gd-DTPA脂质体，进一步评估其淋巴组织靶向增强效果和对良、恶性淋巴结鉴别诊断的作用。
材料和方法
1．Gd-DTPA脂质体的制备和表征
1．1制备工艺精密称取一定量二硬脂酰磷脂酰胆碱乙醇胺(DSPC)溶于双蒸水中，置于恒温磁力搅拌器内，在69～72℃恒温条件下超声分散1小时，取一定量Gd-DTPA溶液、蒸馏水和无水乙醇与上述产物混合，获得半透明胶体状物，在室温下过夜，第二天在69～72℃恒温条件下水浴孵化1小时，同时用低流速氮气吹拂液体表面除去有机溶剂无水乙醇。将以上产物与32mltris缓冲液混合。再将以上所得产物置于离心机内，4000rpm离心5次，每次约5分钟。每次离心结束后，将上清液分离、储存，将沉淀物溶于新鲜缓冲液内继续离心以分离未包裹的游离药物。将最后所得沉淀物溶于新鲜缓冲液，存于4℃冰箱内备用。
1．2小单室脂质体粒径及其分布的测定
用Malvern-3000HS激光粒度分析仪测定粒径及其分布：吸取5μ1制备的相应样品，用双蒸水稀释至量杯中。所选激光光源波长为633.0hm，测试温度为25.00±0.05。
1．3小单室脂质体的形态观察
用透射电镜(日立H-600)观察小单室脂质体大体形态。具体操作方法为：1∶4蒸馏水稀释液体，取1滴放在镀膜铜网上，滤纸吸去多余液体，加入2％磷钨酸染30s，干燥后电镜下观察。
1．4交错融合大脂质体粒径及其分布的测定
用激光散射粒度分析仪(PCS)测定粒度及其分布：632nm氦氖激光，5mM，90度扫描，298K。测试结果分别给出体均粒径和多分散度。
1．5交错融合大Gd-DTPA脂质体的形态观察
具体操作方法同小单室脂质体。
1．6 Gd-DTPA脂质体包封率及载药量的测定
取5ml前述离心后所得上清液送交中山大学分析测试中心，采用高频电感耦合等离子发射光谱法(ICP-AES)测定离心液中Gd-DTPA含量，用加入总的Gd-DTPA量减去离心液中的Gd-DTPA含量即得包封量。用包封量除以Gd-DTPA脂质体的重量即得载药量。
2．肝转移瘤Gd-DTPA脂质体磁共振增强扫描研究
2．1实验对象分组
健康成年新西兰白兔18只，体重2.0～2.5kg，采用随机法分为3组：1、0.2mmolGd／kg脂质体组(n=6)；2、0.5mmolGd／kg脂质体组(n=6)；3、游离Gd-DTPA组(n=6)。
2．2肝转移瘤动物模型建立
VX_2荷瘤种兔耳静脉全麻，剥离切除肿瘤，放入生理盐水中漂洗，剪去周围包膜，用细剪剪成1mm~3大小的组织块。模型兔麻醉后腹正中切口，打开腹腔后取出肝脏，用巩膜剪在肝表面的三个不同部位形成约2mm×2mm大小的腔隙，再将3块1mm~3的肿瘤组织块放入腔隙内，2～3周后VX_2肉瘤肝脏转移瘤模型建成。
2．3超声检查任意选择两只模型兔作超声检查。
2．4 MRI扫描方法
2．4．1平扫：MRI扫描(Siemens Magnetom Vision Plus 1.5T)采用头部线圈，冠状及横断面扫描，扫描参数为T_1WI(TR／TE=500ms／15ms)。另外任意选择两只新西兰白兔尝试用不同序列扫描，扫描序列及参数为：脂肪抑制T_1WI(TR／TE=539／14ms，二次采集)，梯度回波序列(GRE)T_1WI(TR／TE=150／14ms，翻转角70°，1次采集)，SE序列双回波(TR／TE=1800ms／70，20ms，二次采集)，快速自旋回波(TSE)T_2WI(TR／TE=4100／90ms，二次采集)。
2．4．2增强扫描：平扫后，各组分别于静注后5min、15min、30min、1h、2h增强扫描，扫描参数为T_1WI(TR／TE=500ms／15ms)。
2．5图像分析
测量增强前及增强后三组实验动物各时间点T_1WI像上每个病灶及肝实质感兴趣区(Region of interest，ROI)的平均信号强度(Signal intensity，SI)、背景噪声(Noise，N)；计算病变与肝脏的对比噪声比(contrast noise ratio，CNR)。
2．6病理组织学检查
所有实验动物在完成MRI增强扫描后，用耳缘静脉注入空气法处死动物，取出肝脏，10％福尔马林浸泡，以2mm的间隔对肝脏标本切开，计数肿瘤的数目并测量肿瘤的大小。常规石蜡包埋，切片后做H-E染色光学显微镜观察。
2．7统计学分析
使用SPSS 10.0软件包，采用重复测量的方差分析对比增强前后各组肝脏病变的CNR是否有显著性差异。多重比较方法用Bonferroni法，P＜0.05认为有显著性差异。
3．组织间隙注射Gd-DTPA脂质体磁共振淋巴成像的实验研究
3．1实验对象分组
健康成年新西兰白兔18只，体重2.0～2.5kg，采用随机法分为3组：1、对照组(n=6)：未经任何处理，用于对照；2、反应性增生淋巴结组(n=6)：用于建立蛋黄乳胶刺激反应性淋巴结增生模型；3、子宫内膜癌转移淋巴结组(n=6)：用于建立子宫内膜癌淋巴结转移模型。
3．2动物模型建立
3．2．1反应性淋巴结增生模型
取2ml蛋黄乳胶，双侧大腿肌肉各注射0.5ml，双侧腰窝皮下各注射0.5ml，3～4天后再重复上述操作一次，3-4天后可进行MRI扫描。
3．2．2子宫内膜癌淋巴结转移模型
VX_2荷瘤种兔耳静脉全麻，剥离切除肿瘤，剪去周围包膜，用细剪剪成1mm~3大小的组织块。模型兔麻醉后腹正中切口，打开腹腔后取出子宫，暴露子宫内膜，用巩膜剪分离子宫内膜，形成约2mm×2mm大小的内膜腔隙，再将1mm~3的肿瘤组织块放入腔隙内，缝合固定。3周后进行MRI扫描。
3．3 MRI扫描
3．3．1平扫：选用头部相控阵线圈，扫描序列及参数为：T_1WI(TR／TE=539／14ms，二次采集)，脂肪抑制T_1WI(TR／TE=539／14ms，二次采集)T_2WI(TR／TE=2234／85ms，二次采集)。
3．3．2增强扫描
在平扫完毕后，兔双侧大腿皮下各注射0.5ml 0.25mmolGd／kg的Gd-DTPA脂质体，局部按摩5分钟，扫描时间点为给药后5分钟、15分钟、30分钟、45分钟、1小时，扫描参数同平扫。
3．4图像评估
3．4．1测量淋巴结大小
在T_1WI像上，测量各组腹膜后淋巴结的短径，以均数±标准差表示，并与淋巴结病理解剖标本的实测值作对照。
3．4．2测量平扫及增强后各组淋巴结信号强度
在不同序列上分别测量平扫、增强后胭窝、腹股沟及腹膜后淋巴结的信号强度，测背景噪声的标准差(Standard deviation of the noise，SD_N)。计算各组淋巴结的信噪比(Signal to noise ratio，SNR)
3．4．3 Gd-DTPA脂质体增强——时间曲线
在T_1WI FS像测量Gd-DTPA脂质体增强后腹膜后反应性增生淋巴结组上各时间点的SNR，计算强化率(Enhancing rate，En％)
3．5病理组织学检查
所有实验动物在完成MRI增强扫描后，用耳缘静脉注入空气法处死动物，仔细暴露腹膜后淋巴结，摘除并测量淋巴结的短径，计算淋巴结数目。然后用10～15％中性福尔马林浸泡、固定，常规石蜡包埋，切片后做H-E染色用于观察淋巴结形态和结构的变化，判别其良恶性。
3．6统计学分析
应用SPSS10.0软件包，采用完全随机设计方差分析(One-Way ANOVA)评价平扫各组淋巴结的大小、平扫各组不同序列淋巴结的SNR、组间各序列图像上Gd-DTPA脂质体增强后30min腹膜后淋巴结SNR是否有显著性差异，多重比较采用Bonferroni检验。P＜0.05认为有显著性差异。
结果
1．Gd-DTPA脂质体的表征
1．1激光粒度分析仪测定的小单室脂质体平均粒径235nm，粒径分布为0.08；激光散射粒度分析仪测定的交错融合脂质体平均粒径2.2μm。
1．2透射电镜观察小单室脂质体呈类圆形，大小较均匀，表面平滑完整，粒子之间无粘连；透射电镜观察交错融合脂质体呈不规则形，表现为多个小单室脂质体互相融合而成。
1．3 Gd-DTPA脂质体的平均包封率为22％，平均载药量为53％。
2．肝转移瘤Gd-DTPA脂质体磁共振增强扫描研究
2．1模型建立及病理表现
18只新西兰白兔肝转移瘤模型全部建立成功，病理检查共发现35个瘤灶，成瘤率65％。瘤结节大小0.8～1.2cm，平均1.1cm。大体标本显示肝转移瘤结节质地较韧，外观为黄白色，稍突出于肝表面，切面为鱼肉状黄白色，未见明显包膜，大癌结节内可见灶性坏死和出血。HE染色低倍镜下可见肝内浸润性癌巢，与肝实质无明显边界，间质分界不清，结缔组织较少，瘤巢边缘分布着被浸润的肝索结构。肿瘤细胞弥散排列，可见纤维间隔。高倍镜下见瘤细胞体积大，形态不规则，呈不规则排列。胞浆丰富，淡红染色。核肥大，其大小及形态各异，染色浓淡不均，核分裂像多见。间质内可见淋巴细胞、浆细胞浸润。
2．2 MR平扫时不同序列表现
SE序列T_1WI上，肝癌表现为类圆形或不规则低信号或低等混杂信号，坏死表现为更低信号。SE序列FS T_1WI上，肝实质信号相对升高，病变信号亦同时升高，病变肝脏对比度较SE序列T_1WI无明显差别。梯度回波序列(GRE)T_1WI上，病灶表现为低信号，但病灶内部结构及边界显示均较SE序列T_1WI模糊。快速自旋回波(TSE)T_2WI上，病灶表现为高信号，病灶边界显示较模糊。SE双回波T_2WI病灶显示较快速自旋回波(TSE)T_2WI更为模糊，PDWI像上病灶对比差。
2．3 Gd-DTPA脂质体增强后肝转移瘤病灶表现
静脉注射脂质体后，肝脏病灶对比噪声比(CNR)开始上升，至1h达到高峰，随后即开始下降，增强后5个时间点CNR与增强前相比有显著性差异(F=2100.395，P=0.000)。0.5mmolGd／kg脂质体组较0.2mm01Gd／kg脂质体组病灶CNR升高幅度较大，但高峰值均出现在1h左右。静脉注射游离Gd-DTPA后，肝脏病灶CNR下降，增强后5个时间点CNR与增强前相比有显著性差异(F=770.826，P=0.000)。
重复测量方差分析结果显示，不同组问差异有统计学意义(F=17496.40，P=0.000)，不同时间点之间有差异(F=3884.304，P=0.000)，二者有交互效应(F=1645.431，P=0.000)，说明随着时间的变化，三组各时间点肝脏病灶CNR变化趋势不同。
3．组织间隙注射Gd-DTPA脂质体磁共振淋巴成像的实验研究
3．1平扫各组腹膜后淋巴结的大小
完全随机设计方差分析结果显示，三组平扫T_1WI短径有差异(F=31764.21，P=0.000)，病理测量亦有差异(F=18052.71，P=0.000)。多重比较Bonferroni检验结果显示，正常组分别与反应性增生组和转移组比较，平扫T_1WI短径测量P值均＜0.001；病理测量P值均＜0.001。反应性增生组和转移组：多重比较Bonferroni检验结果显示，两组之间平扫T_1WI短径测量无显著性差异(P值＞0.05)，病理测量亦无显著性差异(P值＞0.05)。
3．2各组腹膜后淋巴结平扫信号特点及SNR对比
在T_1WI像上，相对于肌肉及脂肪，各组淋巴结均呈低信号，在T_1WI FS像上，相对于肌肉，各组淋巴结呈等或高信号，在T_2WI像上，相对于肌肉，各组淋巴结均呈高信号。
3．3组织间隙注射Gd-DTPA脂质体后各组淋巴结MRI表现
正常组和反应性增生淋巴结组：组织间隙注射Gd-DTPA脂质体后，与平扫相比，胭窝、腹股沟、腹膜后淋巴结在T_1WI、T_1WI FS上表现为随时间推移的逐渐强化，其中胭窝和腹股沟淋巴结在15min时达到最大强化，腹膜后淋巴结在30min时达到最大强化，随后强化程度下降。
子宫内膜癌转移性淋巴结组：组织间隙注射Gd-DTPA脂质体后，与平扫相比，腹膜后淋巴结在T_1WI、T_1WI FS上表现为不强化或轻度强化。
完全随机设计方差分析结果显示，Gd-DTPA脂质体增强后30min子宫内膜癌腹膜后转移性淋巴结组的SNR在T_1WI和T_1WI FS上小于反应性增生组和正常组(P值均＜0.001)。多重比较Bonferroni检验结果显示，转移组分别与反应性增生组和正常组比较，T_1WI像上SNR比较P值均＜0.001；T_1WI FS像上SNR比较P值均＜0.001。反应性增生组和正常组比较，T_1WI像上SNR比较P=0.111，T_1WI FS像上SNR比较P=0.168，P值均＞0.05，组对比无显著性差异。
3．4腹膜后反应性淋巴结脂质体Gd-DTPA增强—时间曲线
组织间隙注射脂质体Gd-DTPA后，在T_1WI FS像上，腹膜后反应性增生淋巴结第5min强化率为14％，随后迅速升高，第15min达70％，第30min达144％，第45min强化率降低，达87％，1h强化率为20％。
3．5病理表现肿瘤转移性淋巴结：淋巴结的结构呈不同程度破坏，其内可见大量核大而且深染的肿瘤细胞浸润，呈铺路状排列，不形成癌巢。
结论
1．采用乙醇诱导法制备交错融合脂质体的方法是可行的。
2．交错融合脂质体的粒径为2.2um，其包封率及载药量分别为22％、53％，大体形态呈类圆形。
3．采用标准SE序列的T1WI作为兔肝转移瘤模型增强前扫描序列，组织对比度较好，图像质量好。
4．Gd-DTPA脂质体静脉注射可使肝转移瘤动物模型病灶肝脏对比噪声比升高，是提高肝转移瘤检出率的优良载体。
5．Gd-DTPA脂质体具有良好的缓释特点。
6．蛋黄乳胶法建立兔腘窝、腹股沟、腹膜后三组相连续的反应性淋巴结增生模型；兔子宫内膜分离、VX_2组织块包膜法建立兔子宫内膜癌并腹膜后淋巴结转移模型。以上两种模型与人类淋巴结增生和转移模型的MR表现相似，是研究新型淋巴造影对比剂检测恶性淋巴结效能的理想模型。
7．增生组和转移组腹膜后淋巴结大于未处理组者。
8．MRI平扫难以准确区分反应性增生淋巴结和肿瘤转移性淋巴结。
9．组织间隙注射Gd-DTPA脂质体后，反应性增生淋巴结兔模型胭窝、腹股沟淋巴结最大强化峰值在15min左右，腹膜后淋巴结最大强化峰值在30min左右。
10．组织间隙注射Gd-DTPA脂质体后，反应性增生淋巴结与肿瘤转移性淋巴结表现出不同的强化特征，可以用来鉴别良、恶性淋巴结。
Objective
1. To prepare liposomal Gd-DTPA and measurc particle size and distribution;The morphology of the particle was cxamined by transmission electron microscopy.
2. Compared to Gd-DTPA,to investigate the cffect of intravenous injection of liposomal Gd-DTPA as MR specific contrast agents imaging for hepatic mctastascs aftcr Devclopping rabbit model of hcpatic metastascs
3. To further evaluate the effect of liposomal Gd-DTPA targetcd lymphatic tissue and the value of the MR lymphography to differentiate mctastascs from bcnigh lymph nodes after interstitial administration.Reactive hypcrplasia lymph nodes and metastatic lymph nodes were developed.
Materials and Methods
1. Preparation and exosyndrome of liposomal Gd-DTPA
1.1 Preparation
small unilamellar vesicles were prepared by combining 0.92 g of distearoylphosphatidylcholinc (DSPC) with 23 mL of distilled watcr.The mixture underwent sonication for 1 hour.A contrast material-ethanol mixture was prepared by combining Gd-DTPA,distillcd water(6.7 mL),and ethanol(16.3mL).The mixture was combined with cqual amounts of the previously prepared small unilameller vesicles to produce a translucent gel.The gel was allowed to stand at room temperature overnight. The mixture was then incubated in a hot-water bath for approximately I hour and sparged with a slow stream of nitrogen gas.The resulting material was allowed to cool to room temperature and mixed with 100 mL of tris(hydroxymethyl)aminomethane (Tris) buffer (pH, 7.1; 20 mmol/L Tris, 0.1 g/L sodium calcium edetic acid in 0.9% saline solution).To isolate the liposomes,the mixture was centrifuged at 4,000 rpm for about 5 minutes.The supernatant was removed and saved, and the liposome-containing pellet was resuspended in fresh buffer. This isolation procedure was repeated until the total counts in the wash were less than 1% of the total added to the original mixture (the washing procedure was usually repeated five times).The final, washed liposome suspension can be brought to any desired volume with buffer.
1.2 Small unilamellar vesicles size and size distribution measured
The size and size distribution were measured using dynamic light scattering (a Malvern Zetasizer 30000HS). A sample volume of 5ul was diluted with distilled water then measured under 633nm laser and a temperature of 25.00±0.05.
1.3 The shape morphology of Small unilamellar vesicles
The morphology of Gd-PBCA-NP was examined by transmission electron microscopy. A little of the diaylsed solution was dilluted with distilled water (1:4) and applied to metallic sample plate following negative staining with sodium phosphotungstate solution. The sample was freeze-dried and examined by transmission electron microscopy
1.4 IFvs size and size distribution measured
1.5 The shape morphology of IFvs
1.6 Measureing the encapsulation efficiency and loading capacity of Liposomal Gd-DTPA
A sample volume of 5ml was sent to analyze (Zhongshan University Analytical Center, Guangzhou).The concentration of Gd-DTPA in dialysed solution were measured by inductively coupled plasma atomic emission spectrophotometry (ICP-AES).
2. The study of liposomal Gd-DTPA targeted hepatic metastases
2.1 Animals
In this experiment, 18 New Zealand White Rabbits weighted at 2.0～2.5kg were used. All the animals were divided into 3 groups at random:0.2mmolGd/kg liposome group(n=6); 0.5mmolGd/kg liposome group(n=6);Gd-DTPA control group(n=6).All rabbits were buying from the animal center of NanFang hospital.
2.2 The development of models
A certain quantity of fresh tumor tissue was obtained from the tumor-bearing rabbit and was skived to little pieces.Three pieces of tumor tissue were implanted into the different site of the liver.
2.3 Ultrasound
Two animal models were studied by conventional ultrasound
2.4 MR imaging
All MR imaging was performed before and after different enhancement protocols at field strength of 1.5 Tesla MR scanner (Magnetom vision plus 4, VB33A; Siemens, Germany) with use of a head coil for transmission and reception of the signal. MR sequences included Tl-weighted spin echo images (repetition time [TR] mess/echo time [TE] mess, 500/15);Two animals had other sequences included fat suppression(FS)TlWI(539/14)and gradient-echo(GRE)TlWI(150/14,flip angle,70°),double SE(T2-weighted:1800/70;PDWI:1800/20)and T2-weighted fast SE images(4100/90).
Immediately after contrast medium injection, dynamic data of different group were aqcquired at 5min, 15min, 30min, lh, 2h using the Tl-weighted spin echo images.
2.5 MR imaging analysis
Average signal intensity (SI) over region-of-interest (ROI) drawn on hepatic metastases and liver were measured on MR images. Background noise was measured in each image and its ROI was placed adjacent background outside abdomen. Contrast to noise ratio(CNR)of lesions and liver were calculated on all images.
2.6 Pathological examination:
After MRI,the liver were transected into slices with 2.0mm and taken count of the lesions.All liver tissues were fixed in neutral formalin solution and embedded with paraffin and sliced up.These slices were preformed with HE staining.
2.7 Statistical analysis
SPSS 10.0 was used as analyzing software. Repeated measures ANOVA analyzed the difference in CNR of liver metasteses among different groups. P＞0.05 was regarded as no statistical difference; P<0.05 was regarded as statistical significant difference.
3. MR lymphography after interstitial administration of liposomal Gd-DTPA
3.1 Animals
18 New Zealand White Rabbits weighted at 2.0-2.5kg were used. All the animals were divided into 3 groups at random:1.control group(n=6);2.reactive hyperplasia group(n=6);3.endometrial neoplasms-bearing group(n=6).
3.2 The development of models
3.2.1 Reactive hyperplasia model
Each rabbit was injected 0.5ml of egg-yolk emulsion bilaterally in the femoral muscles and subcutaneously in the flank.The injections were repeated 3-4 days later.Magnetic resonance imaging was performed 3-4 days after the second administration.
3.2.2 Endometrial neoplasms-bearing model
A certain quantity of fresh tumor tissue was obtained from the tumor-bearing rabbit and was skived to little pieces,VX2 tumor grafts were established by orthotopic embedding endometrium of rabbits.
3.3 MR imaging
3.3.1 The non-enhanced sequences consisted of TlWI (TR/TE=539/14ms), T_1WI FS(TR/TE=539/14ms) ,T_2WI(TR/TE=2234/85ms).
3.3.2 Liposomal Gd-DTPA(0.25mol Gd/L) were injected into both thighs(0.5ml,each).Following administration,massage was performed at the injection sites for 5min to assist the lymph flow of the anesthetized animals.MR imaging was performed 5,15,30,45min and lh after administration of the agents using the same imaging parameters as for the precontrast images.
3.4 MR imaging analysis
3.4.1 Size of lymph nodes were measured on T1WI
3.4.2 The signal intensity of iliac medial,superficial inguinal and popliteal lymph nodes were measured on different sequences of images before and after contrast enhancement.Background noise(N)was measured in each image.Signal to noise ratio(SNR)were calculated on all images.The enhanced ratio was calculated on T_1WI images after injected liposomal Gd-DTPA in the reactive hyperplasia lymph node group.
3.5 Histopathologic examination
After the completion of MR imaging, all animals were deeply anesthetized and then were sacrificed by exasnguinations. Iliac medial lymph nodes were exposed to determine their positions and then were carefully removed. The size of each lymph node was measured and the amount of lymph nodes was calculated. Removed lymph nodes were fixed and subjected to H-E staining so that benign or malignant lymph node could be assessed.
3.6 Statistical analysis
SPSS 10.0 was used as analyzing software. One-way ANOVA analyzed the difference in size and SNR of lymph nodes on plain images, on liposomal Gd-DTPA enhanced images of lymph nodes between groups which was performed 30min after administration. P＜0.05 was regarded as statistical difference.
Results
1. Exosyndrome of liposomal Gd-DTPA
1.1 The average size of SUVs and IFvs were 235nm and 2.2μm,and the index of size distribution of SUVs was 0.08.
1.2 The morphology of liposomal Gd-DTPA was spherical shape and without adherence between particles observed by transmission electron microscopy.
1.3 The average encapsulation efficiency and loading capacity of liposomal Gd-DTPA were respective 22 %, 51.23 %.
2. The study of liposomal Gd-DTPA targeted hepatic metastases
2.1 The development of models and histopathology
Hepatic metastases were successfully induced in all rabbits in which 35 lesions were founded.The tumor growth rate was 65%. Histopathology show that the implanted tuomrs had imfiltrated into the rabbit liver tissue, similar as the biological features of squamous cell carcinoma being transplanted in other sites of rabbits.
2.2 MR imaging on different sequences before enhanced
On T1WI,hepatic metastases were hypointensity or iso-hypointensity.The signal intensity of the hepatic parenchyma was comparatively higher so that the edge and inside of tumor was clear.On FS T1WI, the signal intensity of the hepatic parenchyma and hepatic metaseses were higher relative to T1WI,so the contrast of lesions were no better comparable to T1WI.
2.3 MR imaging after enhanced
After intravenous injection of liposomal Gd-DTPA, the difference in the CNR of lesions were significant at 5 different times.The CNR of 0.5mmolGd/kg liposome group were higher than that of 0.2mmolGd/kg liposome group.The summit were all lh after enhanced in two groups.After intravenous injection of Gd-DTPA, the difference in the CNR of lesions were significant between non-enhanced images and enhanced images at all 5 different times.After intravenous injection of PBCA-NP, the difference in the signal-to-noise ratios of liver and muscle between before and after enhanced images in all times.There were significant difference in enhanced style between three groups.
3. MR lymphography after interstitial administration of liposomal Gd-DTPA
3.1 On non-enhanced images, the size of iliac medial lymph nodes had significant difference between control groups and reactive,tumor-bearing groups.
3.2 On non-enhanced images ,the signal-to-noise ratios had no significant difference in three sequences of images of three groups,
3.3 On liposomal Gd-DTPA enhanced T_1WI, T_1WI FS images ,the reactive hyperplasia lymph nodes demonstrated obviously homogenous enhancement while metastatic lymph nodes showed heterogeneous or no enhancement, there were significant difference in the signal-to-noise ratios of lymph nodes
3.4 After interstitial administration of liposomal Gd-DTPA ,the signal-to-noise ratios of the reactive hyperplasia iliac medial lymph nodes increased quickly, and the maximal enhancement occurred at 30min.
3.5 Histopathological findings showed that the structure of the metastatic lymph nodes were destroyed at varied extent by tumor tissue.
Conclusions
1. Interdigitation-fusion as a new method for procducing lipod vesicles of high internal volume is feasible.
2. The average size of liposomal Gd-DTPA was 2.2 um and the average encapsulation efficiency and loading capacity of Gd-PBCA-NP were respective22%, 51.23 %.The morphology of liposomal Gd-DTPA was spherical shape.
3. On Tl-weighted spin echo images,the contrast of hepatic metastases was better than that of other sequences.
4. After intravenous injection of liposomal Gd-DTPA, the contrast of hepatic metastases was better than that before enhanced.So liposomal Gd-DTPA can be taken as MRI contrast agents targeted for reticuloendothelial system.
5. Liposomal Gd-DTPA has characteristic of delayed release.
6. The lymph of the two models resemble the MR appearance of hyperplastic and metastatic lymph nodes in humans.Therefore,these models are well suited for testing the efficacy of MR-tomographic techniques in the detection of lymph-node metastasis.
7. Reactive and tumor-bearing iliac medial lymph nodes were larger than control.
8. Plain MR scan can not differentiate metastatic lymph nodes from normal and reactive hyperplasia nodes accurately.
9. After interstitial administration of liposomal Gd-DTPA, the maximal enhancement of the reactive iliac medial hyperplasia lymph nodes occurred at 30min, so MR lymphography may be carried out at this time.
10. Metastatic lymph nodes showed different enhancing characteristics from those of reactive hyperplasia nodes in MR lymphography after interstitial administration of liposomal Gd-DTPA, so they can be differentiated accurately.
1．尝试制备装载Gd-DTPA的脂质体，并对其粒径大小、粒径分布、表面电位进行表征；透射电镜来测定其形态和大小；
2．建立肝转移瘤动物模型，静脉注射Gd-DTPA脂质体，并与游离Gd-DTPA溶液对照，考察网状内皮系统特异性对比剂Gd-DTPA脂质体对提高肝脏转移瘤对比度的价值；
3．建立新西兰白兔腹膜后淋巴结增生模型和子宫内膜癌伴腹膜后淋巴结转移模型，组织间隙注射Gd-DTPA脂质体，进一步评估其淋巴组织靶向增强效果和对良、恶性淋巴结鉴别诊断的作用。
材料和方法
1．Gd-DTPA脂质体的制备和表征
1．1制备工艺精密称取一定量二硬脂酰磷脂酰胆碱乙醇胺(DSPC)溶于双蒸水中，置于恒温磁力搅拌器内，在69～72℃恒温条件下超声分散1小时，取一定量Gd-DTPA溶液、蒸馏水和无水乙醇与上述产物混合，获得半透明胶体状物，在室温下过夜，第二天在69～72℃恒温条件下水浴孵化1小时，同时用低流速氮气吹拂液体表面除去有机溶剂无水乙醇。将以上产物与32mltris缓冲液混合。再将以上所得产物置于离心机内，4000rpm离心5次，每次约5分钟。每次离心结束后，将上清液分离、储存，将沉淀物溶于新鲜缓冲液内继续离心以分离未包裹的游离药物。将最后所得沉淀物溶于新鲜缓冲液，存于4℃冰箱内备用。
1．2小单室脂质体粒径及其分布的测定
用Malvern-3000HS激光粒度分析仪测定粒径及其分布：吸取5μ1制备的相应样品，用双蒸水稀释至量杯中。所选激光光源波长为633.0hm，测试温度为25.00±0.05。
1．3小单室脂质体的形态观察
用透射电镜(日立H-600)观察小单室脂质体大体形态。具体操作方法为：1∶4蒸馏水稀释液体，取1滴放在镀膜铜网上，滤纸吸去多余液体，加入2％磷钨酸染30s，干燥后电镜下观察。
1．4交错融合大脂质体粒径及其分布的测定
用激光散射粒度分析仪(PCS)测定粒度及其分布：632nm氦氖激光，5mM，90度扫描，298K。测试结果分别给出体均粒径和多分散度。
1．5交错融合大Gd-DTPA脂质体的形态观察
具体操作方法同小单室脂质体。
1．6 Gd-DTPA脂质体包封率及载药量的测定
取5ml前述离心后所得上清液送交中山大学分析测试中心，采用高频电感耦合等离子发射光谱法(ICP-AES)测定离心液中Gd-DTPA含量，用加入总的Gd-DTPA量减去离心液中的Gd-DTPA含量即得包封量。用包封量除以Gd-DTPA脂质体的重量即得载药量。
2．肝转移瘤Gd-DTPA脂质体磁共振增强扫描研究
2．1实验对象分组
健康成年新西兰白兔18只，体重2.0～2.5kg，采用随机法分为3组：1、0.2mmolGd／kg脂质体组(n=6)；2、0.5mmolGd／kg脂质体组(n=6)；3、游离Gd-DTPA组(n=6)。
2．2肝转移瘤动物模型建立
VX_2荷瘤种兔耳静脉全麻，剥离切除肿瘤，放入生理盐水中漂洗，剪去周围包膜，用细剪剪成1mm~3大小的组织块。模型兔麻醉后腹正中切口，打开腹腔后取出肝脏，用巩膜剪在肝表面的三个不同部位形成约2mm×2mm大小的腔隙，再将3块1mm~3的肿瘤组织块放入腔隙内，2～3周后VX_2肉瘤肝脏转移瘤模型建成。
2．3超声检查任意选择两只模型兔作超声检查。
2．4 MRI扫描方法
2．4．1平扫：MRI扫描(Siemens Magnetom Vision Plus 1.5T)采用头部线圈，冠状及横断面扫描，扫描参数为T_1WI(TR／TE=500ms／15ms)。另外任意选择两只新西兰白兔尝试用不同序列扫描，扫描序列及参数为：脂肪抑制T_1WI(TR／TE=539／14ms，二次采集)，梯度回波序列(GRE)T_1WI(TR／TE=150／14ms，翻转角70°，1次采集)，SE序列双回波(TR／TE=1800ms／70，20ms，二次采集)，快速自旋回波(TSE)T_2WI(TR／TE=4100／90ms，二次采集)。
2．4．2增强扫描：平扫后，各组分别于静注后5min、15min、30min、1h、2h增强扫描，扫描参数为T_1WI(TR／TE=500ms／15ms)。
2．5图像分析
测量增强前及增强后三组实验动物各时间点T_1WI像上每个病灶及肝实质感兴趣区(Region of interest，ROI)的平均信号强度(Signal intensity，SI)、背景噪声(Noise，N)；计算病变与肝脏的对比噪声比(contrast noise ratio，CNR)。
2．6病理组织学检查
所有实验动物在完成MRI增强扫描后，用耳缘静脉注入空气法处死动物，取出肝脏，10％福尔马林浸泡，以2mm的间隔对肝脏标本切开，计数肿瘤的数目并测量肿瘤的大小。常规石蜡包埋，切片后做H-E染色光学显微镜观察。
2．7统计学分析
使用SPSS 10.0软件包，采用重复测量的方差分析对比增强前后各组肝脏病变的CNR是否有显著性差异。多重比较方法用Bonferroni法，P＜0.05认为有显著性差异。
3．组织间隙注射Gd-DTPA脂质体磁共振淋巴成像的实验研究
3．1实验对象分组
健康成年新西兰白兔18只，体重2.0～2.5kg，采用随机法分为3组：1、对照组(n=6)：未经任何处理，用于对照；2、反应性增生淋巴结组(n=6)：用于建立蛋黄乳胶刺激反应性淋巴结增生模型；3、子宫内膜癌转移淋巴结组(n=6)：用于建立子宫内膜癌淋巴结转移模型。
3．2动物模型建立
3．2．1反应性淋巴结增生模型
取2ml蛋黄乳胶，双侧大腿肌肉各注射0.5ml，双侧腰窝皮下各注射0.5ml，3～4天后再重复上述操作一次，3-4天后可进行MRI扫描。
3．2．2子宫内膜癌淋巴结转移模型
VX_2荷瘤种兔耳静脉全麻，剥离切除肿瘤，剪去周围包膜，用细剪剪成1mm~3大小的组织块。模型兔麻醉后腹正中切口，打开腹腔后取出子宫，暴露子宫内膜，用巩膜剪分离子宫内膜，形成约2mm×2mm大小的内膜腔隙，再将1mm~3的肿瘤组织块放入腔隙内，缝合固定。3周后进行MRI扫描。
3．3 MRI扫描
3．3．1平扫：选用头部相控阵线圈，扫描序列及参数为：T_1WI(TR／TE=539／14ms，二次采集)，脂肪抑制T_1WI(TR／TE=539／14ms，二次采集)T_2WI(TR／TE=2234／85ms，二次采集)。
3．3．2增强扫描
在平扫完毕后，兔双侧大腿皮下各注射0.5ml 0.25mmolGd／kg的Gd-DTPA脂质体，局部按摩5分钟，扫描时间点为给药后5分钟、15分钟、30分钟、45分钟、1小时，扫描参数同平扫。
3．4图像评估
3．4．1测量淋巴结大小
在T_1WI像上，测量各组腹膜后淋巴结的短径，以均数±标准差表示，并与淋巴结病理解剖标本的实测值作对照。
3．4．2测量平扫及增强后各组淋巴结信号强度
在不同序列上分别测量平扫、增强后胭窝、腹股沟及腹膜后淋巴结的信号强度，测背景噪声的标准差(Standard deviation of the noise，SD_N)。计算各组淋巴结的信噪比(Signal to noise ratio，SNR)
3．4．3 Gd-DTPA脂质体增强——时间曲线
在T_1WI FS像测量Gd-DTPA脂质体增强后腹膜后反应性增生淋巴结组上各时间点的SNR，计算强化率(Enhancing rate，En％)
3．5病理组织学检查
所有实验动物在完成MRI增强扫描后，用耳缘静脉注入空气法处死动物，仔细暴露腹膜后淋巴结，摘除并测量淋巴结的短径，计算淋巴结数目。然后用10～15％中性福尔马林浸泡、固定，常规石蜡包埋，切片后做H-E染色用于观察淋巴结形态和结构的变化，判别其良恶性。
3．6统计学分析
应用SPSS10.0软件包，采用完全随机设计方差分析(One-Way ANOVA)评价平扫各组淋巴结的大小、平扫各组不同序列淋巴结的SNR、组间各序列图像上Gd-DTPA脂质体增强后30min腹膜后淋巴结SNR是否有显著性差异，多重比较采用Bonferroni检验。P＜0.05认为有显著性差异。
结果
1．Gd-DTPA脂质体的表征
1．1激光粒度分析仪测定的小单室脂质体平均粒径235nm，粒径分布为0.08；激光散射粒度分析仪测定的交错融合脂质体平均粒径2.2μm。
1．2透射电镜观察小单室脂质体呈类圆形，大小较均匀，表面平滑完整，粒子之间无粘连；透射电镜观察交错融合脂质体呈不规则形，表现为多个小单室脂质体互相融合而成。
1．3 Gd-DTPA脂质体的平均包封率为22％，平均载药量为53％。
2．肝转移瘤Gd-DTPA脂质体磁共振增强扫描研究
2．1模型建立及病理表现
18只新西兰白兔肝转移瘤模型全部建立成功，病理检查共发现35个瘤灶，成瘤率65％。瘤结节大小0.8～1.2cm，平均1.1cm。大体标本显示肝转移瘤结节质地较韧，外观为黄白色，稍突出于肝表面，切面为鱼肉状黄白色，未见明显包膜，大癌结节内可见灶性坏死和出血。HE染色低倍镜下可见肝内浸润性癌巢，与肝实质无明显边界，间质分界不清，结缔组织较少，瘤巢边缘分布着被浸润的肝索结构。肿瘤细胞弥散排列，可见纤维间隔。高倍镜下见瘤细胞体积大，形态不规则，呈不规则排列。胞浆丰富，淡红染色。核肥大，其大小及形态各异，染色浓淡不均，核分裂像多见。间质内可见淋巴细胞、浆细胞浸润。
2．2 MR平扫时不同序列表现
SE序列T_1WI上，肝癌表现为类圆形或不规则低信号或低等混杂信号，坏死表现为更低信号。SE序列FS T_1WI上，肝实质信号相对升高，病变信号亦同时升高，病变肝脏对比度较SE序列T_1WI无明显差别。梯度回波序列(GRE)T_1WI上，病灶表现为低信号，但病灶内部结构及边界显示均较SE序列T_1WI模糊。快速自旋回波(TSE)T_2WI上，病灶表现为高信号，病灶边界显示较模糊。SE双回波T_2WI病灶显示较快速自旋回波(TSE)T_2WI更为模糊，PDWI像上病灶对比差。
2．3 Gd-DTPA脂质体增强后肝转移瘤病灶表现
静脉注射脂质体后，肝脏病灶对比噪声比(CNR)开始上升，至1h达到高峰，随后即开始下降，增强后5个时间点CNR与增强前相比有显著性差异(F=2100.395，P=0.000)。0.5mmolGd／kg脂质体组较0.2mm01Gd／kg脂质体组病灶CNR升高幅度较大，但高峰值均出现在1h左右。静脉注射游离Gd-DTPA后，肝脏病灶CNR下降，增强后5个时间点CNR与增强前相比有显著性差异(F=770.826，P=0.000)。
重复测量方差分析结果显示，不同组问差异有统计学意义(F=17496.40，P=0.000)，不同时间点之间有差异(F=3884.304，P=0.000)，二者有交互效应(F=1645.431，P=0.000)，说明随着时间的变化，三组各时间点肝脏病灶CNR变化趋势不同。
3．组织间隙注射Gd-DTPA脂质体磁共振淋巴成像的实验研究
3．1平扫各组腹膜后淋巴结的大小
完全随机设计方差分析结果显示，三组平扫T_1WI短径有差异(F=31764.21，P=0.000)，病理测量亦有差异(F=18052.71，P=0.000)。多重比较Bonferroni检验结果显示，正常组分别与反应性增生组和转移组比较，平扫T_1WI短径测量P值均＜0.001；病理测量P值均＜0.001。反应性增生组和转移组：多重比较Bonferroni检验结果显示，两组之间平扫T_1WI短径测量无显著性差异(P值＞0.05)，病理测量亦无显著性差异(P值＞0.05)。
3．2各组腹膜后淋巴结平扫信号特点及SNR对比
在T_1WI像上，相对于肌肉及脂肪，各组淋巴结均呈低信号，在T_1WI FS像上，相对于肌肉，各组淋巴结呈等或高信号，在T_2WI像上，相对于肌肉，各组淋巴结均呈高信号。
3．3组织间隙注射Gd-DTPA脂质体后各组淋巴结MRI表现
正常组和反应性增生淋巴结组：组织间隙注射Gd-DTPA脂质体后，与平扫相比，胭窝、腹股沟、腹膜后淋巴结在T_1WI、T_1WI FS上表现为随时间推移的逐渐强化，其中胭窝和腹股沟淋巴结在15min时达到最大强化，腹膜后淋巴结在30min时达到最大强化，随后强化程度下降。
子宫内膜癌转移性淋巴结组：组织间隙注射Gd-DTPA脂质体后，与平扫相比，腹膜后淋巴结在T_1WI、T_1WI FS上表现为不强化或轻度强化。
完全随机设计方差分析结果显示，Gd-DTPA脂质体增强后30min子宫内膜癌腹膜后转移性淋巴结组的SNR在T_1WI和T_1WI FS上小于反应性增生组和正常组(P值均＜0.001)。多重比较Bonferroni检验结果显示，转移组分别与反应性增生组和正常组比较，T_1WI像上SNR比较P值均＜0.001；T_1WI FS像上SNR比较P值均＜0.001。反应性增生组和正常组比较，T_1WI像上SNR比较P=0.111，T_1WI FS像上SNR比较P=0.168，P值均＞0.05，组对比无显著性差异。
3．4腹膜后反应性淋巴结脂质体Gd-DTPA增强—时间曲线
组织间隙注射脂质体Gd-DTPA后，在T_1WI FS像上，腹膜后反应性增生淋巴结第5min强化率为14％，随后迅速升高，第15min达70％，第30min达144％，第45min强化率降低，达87％，1h强化率为20％。
3．5病理表现肿瘤转移性淋巴结：淋巴结的结构呈不同程度破坏，其内可见大量核大而且深染的肿瘤细胞浸润，呈铺路状排列，不形成癌巢。
结论
1．采用乙醇诱导法制备交错融合脂质体的方法是可行的。
2．交错融合脂质体的粒径为2.2um，其包封率及载药量分别为22％、53％，大体形态呈类圆形。
3．采用标准SE序列的T1WI作为兔肝转移瘤模型增强前扫描序列，组织对比度较好，图像质量好。
4．Gd-DTPA脂质体静脉注射可使肝转移瘤动物模型病灶肝脏对比噪声比升高，是提高肝转移瘤检出率的优良载体。
5．Gd-DTPA脂质体具有良好的缓释特点。
6．蛋黄乳胶法建立兔腘窝、腹股沟、腹膜后三组相连续的反应性淋巴结增生模型；兔子宫内膜分离、VX_2组织块包膜法建立兔子宫内膜癌并腹膜后淋巴结转移模型。以上两种模型与人类淋巴结增生和转移模型的MR表现相似，是研究新型淋巴造影对比剂检测恶性淋巴结效能的理想模型。
7．增生组和转移组腹膜后淋巴结大于未处理组者。
8．MRI平扫难以准确区分反应性增生淋巴结和肿瘤转移性淋巴结。
9．组织间隙注射Gd-DTPA脂质体后，反应性增生淋巴结兔模型胭窝、腹股沟淋巴结最大强化峰值在15min左右，腹膜后淋巴结最大强化峰值在30min左右。
10．组织间隙注射Gd-DTPA脂质体后，反应性增生淋巴结与肿瘤转移性淋巴结表现出不同的强化特征，可以用来鉴别良、恶性淋巴结。
Objective
1. To prepare liposomal Gd-DTPA and measurc particle size and distribution;The morphology of the particle was cxamined by transmission electron microscopy.
2. Compared to Gd-DTPA,to investigate the cffect of intravenous injection of liposomal Gd-DTPA as MR specific contrast agents imaging for hepatic mctastascs aftcr Devclopping rabbit model of hcpatic metastascs
3. To further evaluate the effect of liposomal Gd-DTPA targetcd lymphatic tissue and the value of the MR lymphography to differentiate mctastascs from bcnigh lymph nodes after interstitial administration.Reactive hypcrplasia lymph nodes and metastatic lymph nodes were developed.
Materials and Methods
1. Preparation and exosyndrome of liposomal Gd-DTPA
1.1 Preparation
small unilamellar vesicles were prepared by combining 0.92 g of distearoylphosphatidylcholinc (DSPC) with 23 mL of distilled watcr.The mixture underwent sonication for 1 hour.A contrast material-ethanol mixture was prepared by combining Gd-DTPA,distillcd water(6.7 mL),and ethanol(16.3mL).The mixture was combined with cqual amounts of the previously prepared small unilameller vesicles to produce a translucent gel.The gel was allowed to stand at room temperature overnight. The mixture was then incubated in a hot-water bath for approximately I hour and sparged with a slow stream of nitrogen gas.The resulting material was allowed to cool to room temperature and mixed with 100 mL of tris(hydroxymethyl)aminomethane (Tris) buffer (pH, 7.1; 20 mmol/L Tris, 0.1 g/L sodium calcium edetic acid in 0.9% saline solution).To isolate the liposomes,the mixture was centrifuged at 4,000 rpm for about 5 minutes.The supernatant was removed and saved, and the liposome-containing pellet was resuspended in fresh buffer. This isolation procedure was repeated until the total counts in the wash were less than 1% of the total added to the original mixture (the washing procedure was usually repeated five times).The final, washed liposome suspension can be brought to any desired volume with buffer.
1.2 Small unilamellar vesicles size and size distribution measured
The size and size distribution were measured using dynamic light scattering (a Malvern Zetasizer 30000HS). A sample volume of 5ul was diluted with distilled water then measured under 633nm laser and a temperature of 25.00±0.05.
1.3 The shape morphology of Small unilamellar vesicles
The morphology of Gd-PBCA-NP was examined by transmission electron microscopy. A little of the diaylsed solution was dilluted with distilled water (1:4) and applied to metallic sample plate following negative staining with sodium phosphotungstate solution. The sample was freeze-dried and examined by transmission electron microscopy
1.4 IFvs size and size distribution measured
1.5 The shape morphology of IFvs
1.6 Measureing the encapsulation efficiency and loading capacity of Liposomal Gd-DTPA
A sample volume of 5ml was sent to analyze (Zhongshan University Analytical Center, Guangzhou).The concentration of Gd-DTPA in dialysed solution were measured by inductively coupled plasma atomic emission spectrophotometry (ICP-AES).
2. The study of liposomal Gd-DTPA targeted hepatic metastases
2.1 Animals
In this experiment, 18 New Zealand White Rabbits weighted at 2.0～2.5kg were used. All the animals were divided into 3 groups at random:0.2mmolGd/kg liposome group(n=6); 0.5mmolGd/kg liposome group(n=6);Gd-DTPA control group(n=6).All rabbits were buying from the animal center of NanFang hospital.
2.2 The development of models
A certain quantity of fresh tumor tissue was obtained from the tumor-bearing rabbit and was skived to little pieces.Three pieces of tumor tissue were implanted into the different site of the liver.
2.3 Ultrasound
Two animal models were studied by conventional ultrasound
2.4 MR imaging
All MR imaging was performed before and after different enhancement protocols at field strength of 1.5 Tesla MR scanner (Magnetom vision plus 4, VB33A; Siemens, Germany) with use of a head coil for transmission and reception of the signal. MR sequences included Tl-weighted spin echo images (repetition time [TR] mess/echo time [TE] mess, 500/15);Two animals had other sequences included fat suppression(FS)TlWI(539/14)and gradient-echo(GRE)TlWI(150/14,flip angle,70°),double SE(T2-weighted:1800/70;PDWI:1800/20)and T2-weighted fast SE images(4100/90).
Immediately after contrast medium injection, dynamic data of different group were aqcquired at 5min, 15min, 30min, lh, 2h using the Tl-weighted spin echo images.
2.5 MR imaging analysis
Average signal intensity (SI) over region-of-interest (ROI) drawn on hepatic metastases and liver were measured on MR images. Background noise was measured in each image and its ROI was placed adjacent background outside abdomen. Contrast to noise ratio(CNR)of lesions and liver were calculated on all images.
2.6 Pathological examination:
After MRI,the liver were transected into slices with 2.0mm and taken count of the lesions.All liver tissues were fixed in neutral formalin solution and embedded with paraffin and sliced up.These slices were preformed with HE staining.
2.7 Statistical analysis
SPSS 10.0 was used as analyzing software. Repeated measures ANOVA analyzed the difference in CNR of liver metasteses among different groups. P＞0.05 was regarded as no statistical difference; P<0.05 was regarded as statistical significant difference.
3. MR lymphography after interstitial administration of liposomal Gd-DTPA
3.1 Animals
18 New Zealand White Rabbits weighted at 2.0-2.5kg were used. All the animals were divided into 3 groups at random:1.control group(n=6);2.reactive hyperplasia group(n=6);3.endometrial neoplasms-bearing group(n=6).
3.2 The development of models
3.2.1 Reactive hyperplasia model
Each rabbit was injected 0.5ml of egg-yolk emulsion bilaterally in the femoral muscles and subcutaneously in the flank.The injections were repeated 3-4 days later.Magnetic resonance imaging was performed 3-4 days after the second administration.
3.2.2 Endometrial neoplasms-bearing model
A certain quantity of fresh tumor tissue was obtained from the tumor-bearing rabbit and was skived to little pieces,VX2 tumor grafts were established by orthotopic embedding endometrium of rabbits.
3.3 MR imaging
3.3.1 The non-enhanced sequences consisted of TlWI (TR/TE=539/14ms), T_1WI FS(TR/TE=539/14ms) ,T_2WI(TR/TE=2234/85ms).
3.3.2 Liposomal Gd-DTPA(0.25mol Gd/L) were injected into both thighs(0.5ml,each).Following administration,massage was performed at the injection sites for 5min to assist the lymph flow of the anesthetized animals.MR imaging was performed 5,15,30,45min and lh after administration of the agents using the same imaging parameters as for the precontrast images.
3.4 MR imaging analysis
3.4.1 Size of lymph nodes were measured on T1WI
3.4.2 The signal intensity of iliac medial,superficial inguinal and popliteal lymph nodes were measured on different sequences of images before and after contrast enhancement.Background noise(N)was measured in each image.Signal to noise ratio(SNR)were calculated on all images.The enhanced ratio was calculated on T_1WI images after injected liposomal Gd-DTPA in the reactive hyperplasia lymph node group.
3.5 Histopathologic examination
After the completion of MR imaging, all animals were deeply anesthetized and then were sacrificed by exasnguinations. Iliac medial lymph nodes were exposed to determine their positions and then were carefully removed. The size of each lymph node was measured and the amount of lymph nodes was calculated. Removed lymph nodes were fixed and subjected to H-E staining so that benign or malignant lymph node could be assessed.
3.6 Statistical analysis
SPSS 10.0 was used as analyzing software. One-way ANOVA analyzed the difference in size and SNR of lymph nodes on plain images, on liposomal Gd-DTPA enhanced images of lymph nodes between groups which was performed 30min after administration. P＜0.05 was regarded as statistical difference.
Results
1. Exosyndrome of liposomal Gd-DTPA
1.1 The average size of SUVs and IFvs were 235nm and 2.2μm,and the index of size distribution of SUVs was 0.08.
1.2 The morphology of liposomal Gd-DTPA was spherical shape and without adherence between particles observed by transmission electron microscopy.
1.3 The average encapsulation efficiency and loading capacity of liposomal Gd-DTPA were respective 22 %, 51.23 %.
2. The study of liposomal Gd-DTPA targeted hepatic metastases
2.1 The development of models and histopathology
Hepatic metastases were successfully induced in all rabbits in which 35 lesions were founded.The tumor growth rate was 65%. Histopathology show that the implanted tuomrs had imfiltrated into the rabbit liver tissue, similar as the biological features of squamous cell carcinoma being transplanted in other sites of rabbits.
2.2 MR imaging on different sequences before enhanced
On T1WI,hepatic metastases were hypointensity or iso-hypointensity.The signal intensity of the hepatic parenchyma was comparatively higher so that the edge and inside of tumor was clear.On FS T1WI, the signal intensity of the hepatic parenchyma and hepatic metaseses were higher relative to T1WI,so the contrast of lesions were no better comparable to T1WI.
2.3 MR imaging after enhanced
After intravenous injection of liposomal Gd-DTPA, the difference in the CNR of lesions were significant at 5 different times.The CNR of 0.5mmolGd/kg liposome group were higher than that of 0.2mmolGd/kg liposome group.The summit were all lh after enhanced in two groups.After intravenous injection of Gd-DTPA, the difference in the CNR of lesions were significant between non-enhanced images and enhanced images at all 5 different times.After intravenous injection of PBCA-NP, the difference in the signal-to-noise ratios of liver and muscle between before and after enhanced images in all times.There were significant difference in enhanced style between three groups.
3. MR lymphography after interstitial administration of liposomal Gd-DTPA
3.1 On non-enhanced images, the size of iliac medial lymph nodes had significant difference between control groups and reactive,tumor-bearing groups.
3.2 On non-enhanced images ,the signal-to-noise ratios had no significant difference in three sequences of images of three groups,
3.3 On liposomal Gd-DTPA enhanced T_1WI, T_1WI FS images ,the reactive hyperplasia lymph nodes demonstrated obviously homogenous enhancement while metastatic lymph nodes showed heterogeneous or no enhancement, there were significant difference in the signal-to-noise ratios of lymph nodes
3.4 After interstitial administration of liposomal Gd-DTPA ,the signal-to-noise ratios of the reactive hyperplasia iliac medial lymph nodes increased quickly, and the maximal enhancement occurred at 30min.
3.5 Histopathological findings showed that the structure of the metastatic lymph nodes were destroyed at varied extent by tumor tissue.
Conclusions
1. Interdigitation-fusion as a new method for procducing lipod vesicles of high internal volume is feasible.
2. The average size of liposomal Gd-DTPA was 2.2 um and the average encapsulation efficiency and loading capacity of Gd-PBCA-NP were respective22%, 51.23 %.The morphology of liposomal Gd-DTPA was spherical shape.
3. On Tl-weighted spin echo images,the contrast of hepatic metastases was better than that of other sequences.
4. After intravenous injection of liposomal Gd-DTPA, the contrast of hepatic metastases was better than that before enhanced.So liposomal Gd-DTPA can be taken as MRI contrast agents targeted for reticuloendothelial system.
5. Liposomal Gd-DTPA has characteristic of delayed release.
6. The lymph of the two models resemble the MR appearance of hyperplastic and metastatic lymph nodes in humans.Therefore,these models are well suited for testing the efficacy of MR-tomographic techniques in the detection of lymph-node metastasis.
7. Reactive and tumor-bearing iliac medial lymph nodes were larger than control.
8. Plain MR scan can not differentiate metastatic lymph nodes from normal and reactive hyperplasia nodes accurately.
9. After interstitial administration of liposomal Gd-DTPA, the maximal enhancement of the reactive iliac medial hyperplasia lymph nodes occurred at 30min, so MR lymphography may be carried out at this time.
10. Metastatic lymph nodes showed different enhancing characteristics from those of reactive hyperplasia nodes in MR lymphography after interstitial administration of liposomal Gd-DTPA, so they can be differentiated accurately.
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36 Shahbazi-Gahrouei D, Williams M, Rizvi S, et al. In vivo studies of Gd-DTPA-monoclonal antibody and Gd-porphyrins: Potential magnetic resonance imaging contrast agents for melanoma. J Magn Reson Imaging 2001,14(2): 169-174
37 Artemov D. Molecular magnetic resonance imaging with targeted contrast agents. J Cell Biochem. 2003, 90(3):518-524
38 Sipkins DA, Cheresh DA, Kazemi MR, et al. Detection of tumor angiogenesis in vivo by alphaVbeta3-targeted magnetic resonance imaging. Nat. Med., 1998, 4(5): 623-626
39 Sipkins DA, Gijbels K, Tropper FD, et al. ICAM-1 expression in autoimmune encephalitis visualized using magnetic resonance imaging. J Neuroimmunol. 2000, 104(1):1-9
40 Eavarone DA, Yu X, Bellamkonda RV. Targeted drug delivery to C6 glioma by transferrin-coupled liposomes. J Biomed Mater Res 2000, 51(1): 10-14
41 Matsukawa S, Yamamoto M, Ichinose K,et al. Selective uptake by cancer cells of liposomes coated with polysaccharides bearing 1-aminolactose. Anticancer Res 2000, 20(4):2339-2344
42 Wagner S.Benigh lymph node hyperplasia and lymph node metastases in rabbits.Invest.Radiol 1994,29:364-371
43 Tanoura T,Bernas M,Dakarzanli A,et al.MR lymphography with iron oxide compound AMI-227:studies in ferrets with filariasis.AJR 1992;159:875-881.
44 Tjernberg B.An animal study on the diagnosis of VX2 carcinoma and inflammation.Acta Radiol 1962;suppl no.214.
45 Fischman AJ,Ruvin RN,Khaw BA,et al.Detection of acute inflammation with In labeled nonspecific polyclonal IgG.Semin NuclMed 1988;18:335-344.
46 Dresser DW.Immunization of experimental animals.In:Weir DM,ed.Handbook of experimental immunology.Oxford,England:Blackwell Scientific Publications;1986:8.1-8.20.
47 Christofferson RH, Skoldenberg EG, Nillson BO. Tumor growth in a defined microcirculation.MPMIS, 1997,105(6):487-49.
48 Kyotani S, Nishioka Y, Kusunose M, et al.A study of cis-Dianminedichloroplatinum( II )suppositories for the treatment of rabbit uterine endometrial carcinoma.Bio Pharm Bull,1993,16(1):55-58
49. Bellin MF, Roy C, Kinkel K et al (Lymph node metastases: safety and effectiveness of MR imaging with ultrasmall superparamagnetic iron oxide particles-initial clinical experience. Radiology. 1998;207:799-808
50. Vassallo P, Matei C, Heston WDW, et al. Characterization of reactive versus tumor-bearing lymph nodes with interstitial magnetic resonance lymphography in an animal model. Invest Radiol 1995;30:706-711.
51. Sigal R, Vogl T, Casselman J et al Lymph node metastases from head and neck squamous cell carcinoma:MR imaging with ultrasmall superparamagnetic iron oxide particles (Sinerem MR)-results of a phase-Ill multicenter clinical trial. Eur Radiol 2002;12:1104-1113
52. Harisinghani MG, Barentsz J, Hahn PF et al. Noninvasive detection of clinically occult lymph-node metastases in prostate cancer. N Engl J Med 2003;348:2491-2499
53. Michel SC, Keller TM, Frohlich JM,et al. Preoperative breast cancer staging: MR imaging of the axilla with ultrasmall superparamagnetic iron oxide enhancement. Radiology 2002;225:527-536
54. Harisinghani MG, Saini S, Slater GJ, et al. MR imaging of pelvic lymph nodes in primary pelvic carcinoma with ultrasmall superparamagnetic iron oxide(Combidex): preliminary observations.J Magn Reson Imaging 1997;7:161-163
55. Misselwitz B, Platzek J, Raduchel B, et al. Gadofluorine 8: initial experience with a new contrast medium for interstitial MR lymphography. MAGMA. 1999;8:190-195.
56. Ruehm SG, Corot C, Debatin JF Interstitial MR lymphography with a conventional extracellular gadoliniumbased agent: assessment in rabbits.Radiology 2001;218:664-669
57. Bellin MF, Vasile M, Morel-Precetti S.Currently used non-specific extracellular MR contrast media.Eur Radiol 2003;12:2688-2698
58. Ruehm SG, Schroeder T, Debatin JF. Interstitial MR lymphography with gadoterate meglumine: initial experience in humans. Radiology 2001; 220:816-821
59. Christoph U. Herborn, Florian M, et al.Assessment of Normal, Inflammatory, and Tumor-Bearing Lymph Nodes With Contrast-Enhanced Interstitial Magnetic Resonance Lymphography: Preliminary Results in Rabbits. J Magn Reson Imaging 2003;18:328-335
60. Misselwitz B, Platzek J, Weinmann HJ, et al. Early MR Lymphography with Gadofluorine M in RabbitSc Radiology 2004;231:682-688
61. Harika L, Weissleder R, Poss K, et al. Macromolecular intravenous contrast agent for MR lymphography. characterization and efficacy studies. Radiology 1996;198:365-370.
62. Harika L, Weissleder R, Poss K, et al. MR lymphography with a lymphotropic T1-type MR contrast agent: Gd-DTPA-PGM. Magn Reson Med 1995; 33:88-92.
63. Misselwitz B, Schmitt-Willich H, Ebert W, et al. Pharmacokinetics of Gadomer-17, a new dendritic magnetic resonance contrast agent. MAGMA 2001;12:128-134.
[1] Recht A, Houlihan MJ. Axillary lymph nodes and breast cancer: a review. Cancer 1995;76:1491-512.
[2] Hricak H. Imaging of the genitourinary system. Acad Radiol 1995;2:S159-60.
[3] Anzai Y, Brunberg JA, Lufkin RB. Imaging of nodal metastases in the head and neck. J Magn Reson Imaging 1997;7:774-83.
[4] The American Thoracic Society and The European Respiratory Society.Pretreatment evaluation of non-small-cell lung cancer. Am J Respir Crit Care Med 1997; 156:320-332.
[5] Gershenwald J, Thompson W, Mansfield P, et al. Multi-institutional melanoma lymphatic mapping experience: the prognostic value of sentinel lymph node status in 612 stage Ⅰ or Ⅱ melanoma patients.J Clin Oncol 1999;17:976-83.
[6] Laissy J-P, Gay-Depassier P, Soyer P, et al. Enlarged media stinallymph nodes in bronchogenic carcinoma: assessment with dynamic contrast-enhanced MR imaging. Radiology 1994;191:263-7.
[7] van den Brekel MW. Lymph node metastases: CT and MRI. Eur J Radiol 2000;33:230-8.
[8] Murray AD, Staff RT, Redpath TW, et al. Dynamic contrast enhanced MRI of the axilla in women with breast cancer: comparison with pathology of excised nodes. Br J Radiol 2002;75:220-8.
[9] Luciani A, Dao TH, Lapeyre M, et al. Simultaneous bilateral breast and high-resolution axillary MRI of patients with breast cancer: preliminary results. Am J Roentgenol 2004; 182:1059-67.
[10] Harika L, Weissleder R, Poss K, Papisov MI. Macromolecular intravenouscontrast agent for MR lymphography: characterization and efficacy studies. Radiology 1996;198:365-70.
[11] Moghimi SM, Bonnemain B. Subcutaneous and intravenous delivery of diagnostic agents to the lymphatic system: applications in lymphoscintigraphy andindirect lymphography. Adv Drug Deliv Rev 1999;37:295-312.
[12] Steinkamp HJ, Cornehl M, Hosten N, Pegios W, Vogl T, Felix R.Cervical lymphadenopathy: ratio of long- to short-axis diameter as a predictor of malignancy. Br J Radiol 1995;68:266-70.
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[19] Vassallo P, Matei C, Heston WDW, McLachlan SJ, Koutcher JA, Castellino RA. Characterization of reactive versus tumor-bearing lymph nodes with interstitial magnetic resonance lymphography in an animal model. Invest Radiol 1995;30:706-11.
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2 Torchilin VP. Polymeric contrast agents for medical imaging. Curr Pharm Biotechnol 2000,1(2): 183-215
3 Yu X, Song SK, Chen J, et al. High -resolution MRI characterization of human thrombus using a novel fibrin-targeted paramagnetic nanoparticle contrast agent. Magn Reson Med, 2000, 44(6):867-872
4 Lanza GM, Yu X, Winter PM, et al. Targeted antiproliferative drug delivery to vascular smooth muscle cells with a magnetic resonance imaging nanoparticle contrast agent:implications for rational therapy of restenosis.Circulation,2002,106(22):2842-2847
5 Kabalka G, Buonocore E, Hubner K, et al. Gadolinium-labeled liposomes: targeted MR contrast agents for the liver and spleen. Radiology, 1987,163(1):255-258
6 Unger EC, Winokur T, MacDougall P, et al. Hepatic metastases: liposomal Gd-DTPA-enhanced MR imaging. Radiology 1989,171(1):81-85
7 Torchilin VP, Papisov MI. Why do poly-ethylene glycol-coated liposomes circulate so long? J Liposome Res, 1994,4(3), 725-739
8 Bulte, JW, de Cuyper M, Despres D, et al. Short- vs. long-circulating magnetoliposomes as bone marrow-seeking MR contrast agents. J Magn Reson Imaging .1999,9(2): 329-335
9 van den Brekel MW. Lymph node metastases: CT and MRI. Eur J Radiol 2000;33:230-8.
10 Steinkamp HJ, Cornehl M, Hosten N, Pegios W, Vogl T, Felix R.Cervical lymphadenopathy: ratio of long- to short-axis diameter as a predictor of malignancy. Br J Radiol 1995;68:266-70.
11 Anzai Y, Brunberg JA, Lufkin RB. Imaging of nodal metastases in the head and neck. J Magn Reson Imaging 1997;7:774-83.
12 The American Thoracic Society and The European Respiratory Society.Pretreatment evaluation of non-small-cell lung cancer. Am J Respir Crit Care Med 1997; 156:320-332.
13 Taupitz M, Wagner S, Hamm B. Kontrastmittel f(?)ur die magnetresonanztomographische Lymphknotendiagnostik (MRLymphographie).Radiologe 1996;36:134-40.
14 Vassallo P, Matei C, Heston WDW, McLachlan SJ, Koutcher JA,Castellino RA. AMI-227-enhanced MR lymphography: usefulness for differentiating reactive from tumor-bearing lymph nodes. Radiology 1994;193:501-6.
15 Weissleder R, Elizondo G, Wittenberg J, Lee AS, Josephson L, Brady TJ. Ultrasmall superparamagnetic iron oxide: an intravenous contrast agent for assessing lymph nodes with MR imaging. Radiology 1990;175:494-8.
16 Harika L, Weissleder R, Poss K, Zimmer C, Papisov MI, Brady TJ.MR lymphography with a lymphotropic T1-type MR contrast agent: Gd-DTPA-PGM. MRM 1995;33:88-92.
17 Ryman BE, Jewkes RF, Jeyasingh K, et al. Potential application of liposomes to therapy, Ann. NY Acad. Sci. 1978;308:281-307
18 Misselwitz B, Sachse A, Interstitial MR lymphography using GD-carrying liposomes, Acta Radiol. Suppl. 1997, 412:51-55
19 Fujimoto Y, Okuhata Y, Tyngi S, et al. Magnetic resonance lymphography of profunded lymph nodes with liposomal gadolinium-diethylenetriamine penta-acetic acid, Biol Pharm Bull. 2000, 23(1):97-100
20 Trubetskoy VS, Cannillo JA, Milshteyn A, et al. Controlled delivery of Gd-containing liposomes to lymph nodes: surface modification may enhance MRI contrast properties, Magn. Reson. Imag. 1995, 13(1):31-37.
21 李孟达，罗美珍，张彦娜．子宫内膜癌的预后相关因素的探讨．中华肿瘤杂志，1988，10：73-75．
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23 连利娟，主编．林巧稚妇科肿瘤学．第2版北京：人民卫生出版社，2001．369-371．
24 Chan J K, Lin YK, Monk BJ, et al. Vaginal hysterectomy as primary treatment of endometrial cancer in medically compromised women. Obstet Gynecol,2001,97 (5Pt 1):7072711.
25 Jobsen JJ, Schutter EM, Meerwaldt J H, et al. Treatment results in women with clinical stage Ⅰ and pathologic stage Ⅱ endometrial carcinoma. Int J Gynecol Cancer, 2001,11:49253.
26 Yalman D, Ozsaran Z, Anacak Y, et al. Postoperative radiotherapy in endometrial carcinoma: analysis of prognostic factors in 440 cases. Eur J Gynaecol Oncol ,2000,21:3112315.
27 Tilcock C, Unger E, Cullis P et al. Liposomal Gd-DTPA: preparation and characterization of relaxivity. Radiology, 1989,171(1):77-80.
28 Kabalka GW, Davis MA, Holmberg E, et al. Gadolinium-labeled liposomes containing amphiphilic Gd-DTPA derivatives of varying chain length: targeted MRI contrast enhancement agents for the liver. Magn Res Imaging, 1991, 9(3):373-377
29 Trubetskoy VS, Torchilin VP. New approaches in the chemical design of Gd-containing liposomes for use in magnetic resonance imaging of lymph nodes. J Liposome Res. 1994, 3(4):961-998
30 AhI PL, Chen L, Perkins WR, et al. Interdigitation-fusion: a new method for producinglipid vesicles of high internal volume. Biochim Biophys Acta 1994; 1195:237-244.
31 Janoff AS, Minchey SR, Perkins WR, et al.Interdigitation-fusion vesicles: a new approach for selective opacification of the RES. Invest Radiol 1991; 26:5167-5168.
32 Seltzer SE, Janoff AS, BIau M, Adams DF,Minchey SR, Boni LT. Biodistribution and imaging characteristics of iotrolan-carrying interdigitation-fusion vesicles. Invest Radiol 1991; 26:5169-5171.
33 Boni LT, Minchey SR, Perkins WR, et al.Curvature dependent induction of the interdigitated gel phase in DPPC vesicles.Biochim Biophys Acta 1993; 1146:247-257.
34 Minchey SR, Perkins WR, Ahl PL, et al.Enhanced computed tomographic images of liver and spleen: role of contrast agents encapsulated within interdigitation-fusion vesicles (IFVS) (abstr). Biophys J 1992; 61:A447.
35 Perkins WR, Minchey SR. AhI PL, Janoff AS. The determination of liposome captured volume. Chem Phys Lipids 1993; 64:197-217.
36 Shahbazi-Gahrouei D, Williams M, Rizvi S, et al. In vivo studies of Gd-DTPA-monoclonal antibody and Gd-porphyrins: Potential magnetic resonance imaging contrast agents for melanoma. J Magn Reson Imaging 2001,14(2): 169-174
37 Artemov D. Molecular magnetic resonance imaging with targeted contrast agents. J Cell Biochem. 2003, 90(3):518-524
38 Sipkins DA, Cheresh DA, Kazemi MR, et al. Detection of tumor angiogenesis in vivo by alphaVbeta3-targeted magnetic resonance imaging. Nat. Med., 1998, 4(5): 623-626
39 Sipkins DA, Gijbels K, Tropper FD, et al. ICAM-1 expression in autoimmune encephalitis visualized using magnetic resonance imaging. J Neuroimmunol. 2000, 104(1):1-9
40 Eavarone DA, Yu X, Bellamkonda RV. Targeted drug delivery to C6 glioma by transferrin-coupled liposomes. J Biomed Mater Res 2000, 51(1): 10-14
41 Matsukawa S, Yamamoto M, Ichinose K,et al. Selective uptake by cancer cells of liposomes coated with polysaccharides bearing 1-aminolactose. Anticancer Res 2000, 20(4):2339-2344
42 Wagner S.Benigh lymph node hyperplasia and lymph node metastases in rabbits.Invest.Radiol 1994,29:364-371
43 Tanoura T,Bernas M,Dakarzanli A,et al.MR lymphography with iron oxide compound AMI-227:studies in ferrets with filariasis.AJR 1992;159:875-881.
44 Tjernberg B.An animal study on the diagnosis of VX2 carcinoma and inflammation.Acta Radiol 1962;suppl no.214.
45 Fischman AJ,Ruvin RN,Khaw BA,et al.Detection of acute inflammation with In labeled nonspecific polyclonal IgG.Semin NuclMed 1988;18:335-344.
46 Dresser DW.Immunization of experimental animals.In:Weir DM,ed.Handbook of experimental immunology.Oxford,England:Blackwell Scientific Publications;1986:8.1-8.20.
47 Christofferson RH, Skoldenberg EG, Nillson BO. Tumor growth in a defined microcirculation.MPMIS, 1997,105(6):487-49.
48 Kyotani S, Nishioka Y, Kusunose M, et al.A study of cis-Dianminedichloroplatinum( II )suppositories for the treatment of rabbit uterine endometrial carcinoma.Bio Pharm Bull,1993,16(1):55-58
49. Bellin MF, Roy C, Kinkel K et al (Lymph node metastases: safety and effectiveness of MR imaging with ultrasmall superparamagnetic iron oxide particles-initial clinical experience. Radiology. 1998;207:799-808
50. Vassallo P, Matei C, Heston WDW, et al. Characterization of reactive versus tumor-bearing lymph nodes with interstitial magnetic resonance lymphography in an animal model. Invest Radiol 1995;30:706-711.
51. Sigal R, Vogl T, Casselman J et al Lymph node metastases from head and neck squamous cell carcinoma:MR imaging with ultrasmall superparamagnetic iron oxide particles (Sinerem MR)-results of a phase-Ill multicenter clinical trial. Eur Radiol 2002;12:1104-1113
52. Harisinghani MG, Barentsz J, Hahn PF et al. Noninvasive detection of clinically occult lymph-node metastases in prostate cancer. N Engl J Med 2003;348:2491-2499
53. Michel SC, Keller TM, Frohlich JM,et al. Preoperative breast cancer staging: MR imaging of the axilla with ultrasmall superparamagnetic iron oxide enhancement. Radiology 2002;225:527-536
54. Harisinghani MG, Saini S, Slater GJ, et al. MR imaging of pelvic lymph nodes in primary pelvic carcinoma with ultrasmall superparamagnetic iron oxide(Combidex): preliminary observations.J Magn Reson Imaging 1997;7:161-163
55. Misselwitz B, Platzek J, Raduchel B, et al. Gadofluorine 8: initial experience with a new contrast medium for interstitial MR lymphography. MAGMA. 1999;8:190-195.
56. Ruehm SG, Corot C, Debatin JF Interstitial MR lymphography with a conventional extracellular gadoliniumbased agent: assessment in rabbits.Radiology 2001;218:664-669
57. Bellin MF, Vasile M, Morel-Precetti S.Currently used non-specific extracellular MR contrast media.Eur Radiol 2003;12:2688-2698
58. Ruehm SG, Schroeder T, Debatin JF. Interstitial MR lymphography with gadoterate meglumine: initial experience in humans. Radiology 2001; 220:816-821
59. Christoph U. Herborn, Florian M, et al.Assessment of Normal, Inflammatory, and Tumor-Bearing Lymph Nodes With Contrast-Enhanced Interstitial Magnetic Resonance Lymphography: Preliminary Results in Rabbits. J Magn Reson Imaging 2003;18:328-335
60. Misselwitz B, Platzek J, Weinmann HJ, et al. Early MR Lymphography with Gadofluorine M in RabbitSc Radiology 2004;231:682-688
61. Harika L, Weissleder R, Poss K, et al. Macromolecular intravenous contrast agent for MR lymphography. characterization and efficacy studies. Radiology 1996;198:365-370.
62. Harika L, Weissleder R, Poss K, et al. MR lymphography with a lymphotropic T1-type MR contrast agent: Gd-DTPA-PGM. Magn Reson Med 1995; 33:88-92.
63. Misselwitz B, Schmitt-Willich H, Ebert W, et al. Pharmacokinetics of Gadomer-17, a new dendritic magnetic resonance contrast agent. MAGMA 2001;12:128-134.
[1] Recht A, Houlihan MJ. Axillary lymph nodes and breast cancer: a review. Cancer 1995;76:1491-512.
[2] Hricak H. Imaging of the genitourinary system. Acad Radiol 1995;2:S159-60.
[3] Anzai Y, Brunberg JA, Lufkin RB. Imaging of nodal metastases in the head and neck. J Magn Reson Imaging 1997;7:774-83.
[4] The American Thoracic Society and The European Respiratory Society.Pretreatment evaluation of non-small-cell lung cancer. Am J Respir Crit Care Med 1997; 156:320-332.
[5] Gershenwald J, Thompson W, Mansfield P, et al. Multi-institutional melanoma lymphatic mapping experience: the prognostic value of sentinel lymph node status in 612 stage Ⅰ or Ⅱ melanoma patients.J Clin Oncol 1999;17:976-83.
[6] Laissy J-P, Gay-Depassier P, Soyer P, et al. Enlarged media stinallymph nodes in bronchogenic carcinoma: assessment with dynamic contrast-enhanced MR imaging. Radiology 1994;191:263-7.
[7] van den Brekel MW. Lymph node metastases: CT and MRI. Eur J Radiol 2000;33:230-8.
[8] Murray AD, Staff RT, Redpath TW, et al. Dynamic contrast enhanced MRI of the axilla in women with breast cancer: comparison with pathology of excised nodes. Br J Radiol 2002;75:220-8.
[9] Luciani A, Dao TH, Lapeyre M, et al. Simultaneous bilateral breast and high-resolution axillary MRI of patients with breast cancer: preliminary results. Am J Roentgenol 2004; 182:1059-67.
[10] Harika L, Weissleder R, Poss K, Papisov MI. Macromolecular intravenouscontrast agent for MR lymphography: characterization and efficacy studies. Radiology 1996;198:365-70.
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