Sentinel lymph node metastasis imaging by using nanoparticle carriers in an animal study

我们提出了一种使用多模态成像来促进前哨淋巴结活检检查的新方法，其中放射性和近红外（NIR）荧光纳米颗粒可以描绘位于深部的前哨淋巴结和荧光淋巴结，同时可视化感兴趣领域的解剖结构。基于这个原因，我们开发了含有tech -99m（Tc-99m）和ICG的聚酰胺胺（PAMAM）涂层二氧化硅纳米颗粒。为了测试这种双模成像系统的可行性和实用性，我们进行了动物实验。通过透射电镜和扫描电镜观察，PAMAM包覆二氧化硅纳米颗粒的平均直径为30-50 nm。采用激光散射粒度分析仪（SALD-7100, Shimadzu, Japan）测量颗粒粒度的分布。在乙醇溶液中分布峰在30 nm处。Tc-99m和icg - sulo - osu的联合标记在每次实验前都经过薄层色谱（TLC）验证。6只雄性Wister大鼠舌粘膜下注射纳米颗粒溶液0.1 ml （7.4 MBq，除1只大鼠外，共3.7 MBq， ICG 1微克）。6只大鼠注射后1小时进行淋巴显影（顺序静态图像，每5分钟一次）。闪烁成像显示颈部Tc-99m积聚增加，注射部位摄取明显增加。在最后的静态图像中，6只大鼠中有4只出现单节摄取增加。完成淋巴显像检查后，对动物实施安乐死。在6只大鼠的颈部切开皮肤，发现19个淋巴结。淋巴结切除后行接触片，3个摄取淋巴结明显增加，3个摄取淋巴结弱。所有解剖颈部均行近红外荧光成像。PDE系统（Hamamatsu Photonics Co, Japan）提供颈部淋巴结的清晰荧光图像，实时显示解剖结构。随机评价荧光强度，6个淋巴结从弱到强，（+）~(+++)，其余淋巴结评价为无荧光。放射性结节增多与荧光结节重合。用γ阱计数仪测定4只大鼠15个切除淋巴结的放射性。放射性比较显示，高荧光强度组（4个淋巴结，平均%；0.109±0.067）与低或无荧光强度组（8个淋巴结，平均%；0.001±0.000）差异较大（p<0.05）。透射电镜显示，淋巴结内巨噬细胞胞浆内可见局部分布的黑色小颗粒。小球形颗粒的出现仅限于巨噬细胞，而不是其他淋巴细胞。虽然需要进一步的研究来确定这种双成像纳米颗粒的适当灵敏度和安全性，但结果可能预示着在前哨淋巴结活检中，双模态方法可以更好地检测目标淋巴结。少

We propose a new approach using multimodality imaging to facilitate sentinel node biopsy examinations, in which radioactive and near infrared(NIR) fluorescent nanoparticles can depict deep situated sentinel nodes and fluorescent nodes, as well as simultaneously visualize anatomical configurations in the field of interest. For this reason we have developed polyamidoamine(PAMAM) coated silica nanoparticles loaded with technetium-99m(Tc-99m) and ICG. To test the feasibility and utility of this dual-modality imaging system we conducted animal studies. The mean diameter of PAMAM coated silica nanoparticles was 30-50 nm as evaluated by images of transmission electron microscopy and scanning electron microscopy. The distribution of the particle size was measured by a laser scattering particle size analyzer(SALD-7100, Shimadzu, Japan). The peak of the distribution was 30 nm in ethanol solution. The combined labeling with both Tc-99m and ICG-Sulfo-OSu was verified by thin layer chromatography, … More TLC, before each experiment. 0.1 ml of the nanoparticle solution(7.4 MBq, except for one rat ; with 3.7 MBq, and 1 micro-gramm ICG) was submucosally injected in the tongue of six male Wister rats. Lymphoscintigraphy(sequential static images, every 5 minutes) was performed for 6 rats for one hour immediately after injection. Scintigraphic images showed an increased accumulation of Tc-99m in the neck with a markedly increased uptake at the injection site. Single increased nodal uptake was seen in 4 of 6 rats in the final static images. After completion of lymphoscintigraphy, animals were euthanized. As skin incision was made and 19 lymph nodes were identified in the dissected necks of 6 rats. After lymph node excision, contact-radiography was performed which revealed 3 markedly increased uptake nodes and 3 weak uptake nodes. NIR fluorescence imaging was performed on all dissected necks. The PDE system(Hamamatsu Photonics Co, Japan) provided clear fluorescence images of lymph nodes in the neck with anatomical configurations real time. The intensity of fluorescence was arbitrarily evaluated and 6 lymph nodes were weak to strong, (+)～(+++), and other lymph nodes were evaluated as having no fluorescence. Increased radioactive nodes coincided with fluorescent nodes. Radioactivity of 15 excised lymph nodes in 4 rats was assayed with a gamma-well counter. The radioactivity comparison revealed a large difference between the high fluorescence intensity group(4 lymph nodes, mean%; 0.109±0.067) and thelow or no fluorescence intensity group(8 lymph nodes, mean%; 0.001±0.000), (p<0.05). TEM revealed that small black granules were localized and dispersed within the cytoplasm of macrophages in lymph nodes. The appearance of small sphericalgranules was restricted to macrophages but not other lymphatic cells. Although further studies are needed to determine the appropriate sensitivity and safety of thisdual imaging nanoparticles, the results may promise for a better target nodes detection by the dual modality method in sentinel node biopsy. Less