CL-PEG-MnFe_2O_4纳米胶束介导的肿瘤微血管和微淋巴管双重靶向MRI成像
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摘要
背景与目的：
血行转移和淋巴转移是恶性肿瘤常见的转移途径。自Folkman于1971年首次提出肿瘤发生、发展呈血管依赖性以来，肿瘤血管生成和抗肿瘤血管生成治疗一直是肿瘤研究的热点。近年来，随着一系列淋巴管内皮细胞（LECs）标记物的发现，对肿瘤淋巴管生成的研究也逐渐深入到了分子水平。许多肿瘤（尤其是上皮来源的肿瘤），如乳腺癌等，容易同时发生血行转移和淋巴转移。肿瘤微血管密度（MVD）和微淋巴管密度（LVD）与肿瘤的转移和预后密切相关。对于这些肿瘤，只检测其中某一条转移途径，往往达不到综合判断肿瘤是否易于发生远处转移的目的。同时，为了准确客观地评价肿瘤血管生成和淋巴管生成，还必须将肿瘤新生微血管和微淋巴管与正常组织的微血管和微淋巴管区分开来。
Endoglin又名CD105，是内皮细胞膜表达的糖蛋白，是转化生长因子-β（TGF-β）受体复合物的成分之一。它通过参与TGF-β受体的信号转导，调节内皮-间质的信号传递，参与血管生成，在再生组织、炎症及肿瘤组织的新生血管内皮细胞中过表达，并且Endoglin的表达水平与内皮细胞的增殖程度正相关；基于Endoglin染色的肿瘤MVD评估与肿瘤的预后密切相关。早期研究认为Endoglin仅在处于增殖状态的肿瘤组织血管内皮细胞（VECs）高表达，而在正常组织的VECs不表达或低表达，在LECs不表达。近来研究证明Endoglin也是LECs增殖的标记物，它在正常组织LECs不表达或低表达，而在肿瘤新生LECs高表达。因而，若将Endoglin作为分子靶标，应用于肿瘤的磁共振（MR）分子成像，则有望实现肿瘤微血管生成和微淋巴管生成的双重靶向成像。
目前，国内外均未见采用一种MR对比剂实现肿瘤微血管和微淋巴管特异性检测的相关报道。因此，本研究拟通过高温热解法合成超敏感MnFe_2O_4纳米粒，并用PEG-PCL两嵌段聚合物自组装形成高弛豫率的PEG-PCL-MnFe_2O_4纳米胶束，并进一步通过交联反应耦连能与Endoglin特异性结合的多肽CL-1555(氨基酸序列为AHKHVHHVPVRL)制备成CL-PEG-MnFe_2O_4分子探针，通过体外和体内实验证实该分子探针与肿瘤VECs和LECs的特异性结合，阐明靶向Endoglin分子探针介导的肿瘤微血管和微淋巴管MR成像作用，评价MR成像定量分析肿瘤新生血管及淋巴管的可能性和准确性。
研究内容及方法：
1. PEG-PCL-MnFe_2O_4纳米胶束的合成及表征高温热解法合成MnFe_2O_4纳米粒，利用部分末端功能化的PEG两亲性嵌段聚合物包裹MnFe_2O_4纳米粒自组装形成水溶液中单一分散的PEG-PCL-MnFe_2O_4纳米胶束。并采用透射电子显微镜（TEM）、zeta-粒度仪、红外吸收光谱(IR)及电感耦合等离子体原子发射光谱（ICP-AES）对纳米粒及纳米胶束的形态、粒径、分散性、包被物及铁锰构成比进行表征。用磁共振测量纳米胶束的弛豫率。
2. CL-PEG-MnFe_2O_4纳米胶束的构建及体外实验合成能与Endoglin特异性结合的短肽CL-1555，与PEG-PCL-MnFe_2O_4通过交联反应耦联制备靶向Endoglin的CL-PEG-MnFe_2O_4探针。将VECs及LECs与乳腺癌细胞共培养获取肿瘤源性VECs及LECs。以CL-PEG-MnFe_2O_4标记肿瘤源性VECs及LECs，普鲁士蓝染色及荧光显微镜观察探针与内皮细胞的结合情况，透射电子显微镜观察纳米粒进入胞浆的位置，以PEG-PCL-MnFe_2O_4胶束标记肿瘤源性VECs及LECs及CL-PEG-MnFe_2O_4标记人脐静脉内皮细胞（HUVECs）及LECs为对照。并对标记细胞进行MR成像，了解MR成像在检测标记细胞中的价值。CCK-8试剂盒检测CL-PEG-MnFe_2O_4对细胞增殖活性的影响。对标记后的细胞进行传代，观察传代后细胞标记的阳性率，测量传代细胞内锰、铁元素的量，并将每一代细胞进行MR扫描，观察随着细胞传代磁共振信号的变化规律。
3.裸鼠乳腺癌移植瘤模型的建立及MR成像优化采用小关节线圈及专用小动物线圈对裸鼠移植瘤模型进行MR成像，并对成像序列的参数进行优化，以期评价临床常规磁共振在小动物成像中的实用价值。通过尾静脉及球后静脉两种方式静脉注射超顺磁性氧化铁（SPIO）纳米粒，评价两种方法在裸鼠磁性纳米粒静脉给药中的可行性。
4. CL-PEG-MnFe_2O_4应用于裸鼠乳腺癌移植瘤模型的MR分子成像乳腺癌移植瘤模型经球后静脉注射CL-PEG-MnFe_2O_4探针，采用3.0T磁共振扫描仪进行SE T1WI、FSE T2WI、GRE T2*WI及T2mapping成像，以注射PEG-PCL-MnFe_2O_4为对照；扫描结束后，取肿瘤标本进行病理及免疫组化分析，并与MRI图像进行对照，了解CL-PEG-MnFe_2O_4探针对肿瘤新生血管及淋巴管的双重靶向效能。
结果：
1.所合成的MnFe_2O_4纳米粒呈圆形，粒径分布均匀，粒径在11nm左右，zeta-粒度仪结果显示纳米的大小为11.18±1.72nm，具有很好的单分散性。纳米粒的Fe/Mn摩尔浓度比为2.13:1。CL-PEG-MnFe_2O_4纳米胶束呈簇状及团状分布，由十几到几十个纳米粒聚集而成，胶束粒径为52-86nm， zeta平均粒径约78.8±12.4nm。IR显示在1710cm-1处出现一明显的羰基吸收峰。当铁摩尔浓度为：0mmol/L、0.01mmol/L、0.02mmol/L、0.03mmol/L、0.04mmol/L、0.06mmol/L、0.08mmol/L、0.1mmol/L、0.2mmol/L、0.4mmol/L、0.6mmol/L和0.8mmol/L时，随着浓度的增加，在SE T1WI，信号强度先升高后降低；在FSE T2WI，信号强度逐渐下降；在GRE T2*WI，信号强度随浓度的增加显著降低，GRE T2*WI较SE T1WI和FSE T2WI对纳米粒的检测更敏感（P＜0.05）。
2.成功分离脐静脉内皮细胞（HUVECs）。经诱导后的VECs和LECs免疫荧光染色显示，VECs呈CD105阳性，诱导后的LECs在podoplanin和CD105双染下呈绿色（podoplanin）和红色(CD105)荧光,诱导后的VECs和LECs荧光强度强于未诱导的内皮细胞。在相同铁浓度下，CL-PEG-MnFe_2O_4纳米胶束标记的肿瘤源性VECs和LECs的标记率较PEG-PCL-MnFe_2O_4高。在铁浓度为0μg/mL、0.5μg/mL、1μg/mL、2μg/mL、5μg/mL、10μg/mL时，诱导后和未诱导的LECs的标记率分别为0%、26.95±4.38%、62.73±3.07%、82.34±3.67%、100%、100%和0%、15.61±3.42%、36.35±2.26%、52.31±3.23%、87.53±4.62%、100%。在电子显微镜下标记细胞胞膜上、胞浆内及溶酶体可见包含高电子密度的内吞囊泡和细小颗粒；核周部分次级溶酶体破裂，周围胞浆内可见细小颗粒。在MR成像SE T1WI，信号强度呈缓慢升高；在FSE T2WI，信号强度逐渐下降；在GRE T2*WI，信号强度随浓度的增加显著降低。T2mapping显示随着浓度增加T2值逐渐降低，相对于PEG-PCL-MnFe_2O_4纳米胶束标记肿瘤源性VECs及LECs和CL-PEG-MnFe_2O_4纳米胶束标记VECs及LECs，CL-PEG-MnFe_2O_4纳米胶束标记的肿瘤源性VECs及LECs的T2值降低更明显，但随着铁浓度的增加，两者的差异逐渐减小。CCK-8细胞活性检测显示，在浓度小于20μg/mL时，纳米胶束对VECs细胞增殖活性影响不大，当浓度增加到50μg/mL时，细胞活性受到轻度影响，当铁浓度达到100μg/mL，细胞增殖活性受到明显影响，活性细胞数明显减少。标记细胞的普鲁士蓝染色显示，在铁浓度为50μg/mL以下时，细胞生长良好，细胞形态无明显改变，当铁浓度为100μg/mL时，活细胞数明显减少，细胞出现皱缩。随着标记细胞的传代，标记阳性率逐渐降低，在T2WI，磁共振信号逐渐升高；在T1WI，P1代呈低信号，P2、P3代呈稍高信号，P4代在T1WI、T2WI均呈等信号，ICP-AES显示随着细胞的传代，铁元素迅速减少，而锰元素减少较铁慢，Mn/Fe摩尔浓度比逐渐升高。
3.通过细胞悬液裸鼠皮下接种的方法建立乳腺癌动物模型操作简单、成瘤率为100%，肿瘤潜伏期约1-2周，所形成的肿瘤接近人体同类肿瘤本身的特性。采用小动物专用线圈所获得图像的信噪比、分辨率及对比度较关节小线圈高，图像信号显示更均匀。两周以内的移植瘤大小在1cm左右，肿瘤信号均匀，在T1WI呈稍低信号，T2WI呈较高信号，边界较清晰，可见包膜，与邻近结构无粘连；肿瘤生长三周过后，肿瘤内出现囊变、坏死，坏死区在T1WI呈低信号，T2WI呈高信号。球后静脉穿刺方法简单、易行，通过球后静脉注射SPIO后肝脏内纳米粒的蓄积符合纳米粒在血浆中的清除变化规律，球后静脉注射纳米粒肝脏的信号强度变化率较尾静脉注射大。
4.经球后静脉注射靶向探针CL-PEG-MnFe_2O_4后即刻及5min，肿瘤周围区增强程度高于中央区；注射CL-PEG-MnFe_2O_4后1h，肿瘤周边区仍呈点状或片状增强；而注射PEG-PCL-MnFe_2O_4后1h，肿瘤信号与增强前肿瘤信号基本相同。普鲁士蓝染色显示在肿瘤周边区可见大量蓝染颗粒，CD105、CD34及Podoplanin免疫组化染色显示相应区域局部血管及淋巴管丰富，与MRI图像肿瘤增强区相对应。靶向增强组在注射对比剂后即刻信号降低，最大信号强度变化率约为16%，之后肿瘤信号强度缓慢回升，1h后信号变化不明显，信号强度约为峰值信号强度的48%，时间信号强度曲线（TIC）呈下降-上升-平台型。对照组注射对比剂即刻，肿瘤信号强度降低，随时间延长，信号强度逐渐回升；30min时，信号强度接近注射前水平，此时，肿瘤信号与增强前无明显差异，TIC呈下降-上升型。
结论：
1.通过高温热解法能够合成高品质的MnFe_2O_4纳米粒。采用两亲嵌段聚合物自组装形成的PEG-PCL-MnFe_2O_4纳米胶束，具有较强的T2弛豫效能，在相同条件下较PEG-PCL-Fe3O4弛豫性能更高，是较敏感的T2WI对比剂。
2. HUVECs获取方便、分离方法简单、一次获取细胞量大，是研究内皮细胞特性可靠的细胞模型。通过内皮细胞与肿瘤细胞共培养能够使内皮细胞向肿瘤源性内皮细胞分化，是获取肿瘤源性内皮细胞的可靠方法。
3．细胞悬液裸鼠皮下接种的方法建立乳腺癌动物模型操作简单、成瘤率高，所构建的乳腺癌肿瘤是研究血管及淋巴管生成可靠的动物模型。
4.球后静脉穿刺较尾静脉穿刺更简单易行，是静脉注射磁性纳米粒可靠的给药途径。
5.耦连靶向Endoglin的结合肽后所构建的CL-PEG-MnFe_2O_4纳米胶束能够与肿瘤源性的VECs及LECs特异性结合，静脉注射后能够结合到乳腺癌新生血管及淋巴管内皮，并可以通过磁共振进行检测。在一定的浓度范围内对细胞无明显毒性，为应用MR特异性显示肿瘤新生血管和淋巴管提供了可能。
6. MnFe_2O_4纳米粒经细胞代谢后释放出顺磁性的锰离子（Mn2+），并能在细胞内较长时间停留，在一定浓度下可以通过T1WI成像进行检测，有望拓展MnFe_2O_4纳米粒在MR双对比领域的应用前景。
Background and Objective：
Hematogenous and lymphatic metastasis is the most common form of metastasis inpatients with malignant tumor. The angiogenesis and anti-angiogenesis therapy is alwaysthe hot point in cancer research since Folkman first proposed the hypothesis of tumordevelopment was dependent vascular in1971. In recent years, lymphangiogenesisgradually in depth study at the molecular level with a series of lymphatic endothelial cells(LECs) markers found on tumor. Many tumors (especially tumors of epithelial origin, suchas breast cancer, et al.) likely to simultaneously led to hematogenous and lymphaticmetastasis. Microvessel density (MVD) and lymphatic vessel density (LVD) are closelyrelated to the metastasis and prognosis of tumor. So, it is difficult to determine whether thetumor has occurred metastasis or not if we only focus on one metastatic pathway. At thesame time, In order to evaluate the angiogenesis and lymphangiogenesis accurately, wemust differentiate the tumor neoangiogenesis and neonatal lymphatic vessels from themicrovessels in the normal tissue.
Endoglin, also known as CD105, is a glycoprotein expressed in endothelial cellmembrane, is one of the transforming growth factor-β (TGF-β) receptor complex. Endoglinregulate the signal transduction between endothelial cell and interstitial tissue through theTGF-β, and promote the angiogenesis. Endoglin overexpressed in neonatal vascularendothelial cells in regenerated tissue, inflammation and tumor tissue. The expression levelsand degree of Endoglin is closely related to the endothelial cell proliferation. The MVDbased on Endoglin staining is closely related to the tumor prognosis. Endoglin is also theproliferation marker of LECs, which is overexpressed in neonatal lymphatic vessel, but notexpressed in the LECs of normal tissue. Thus, it is expected to achieve dual targeting imaging of breast cancer angiogenesis and lymphangiogenesis if Endoglin was used as amolecular target in magnetic resonance (MR) molecular imaging.
To our knowledge, there is no relevant report on simultaneous detection of tumormicrovessel and microlymphatic vessel using a MR contrast agent. In this study, MnFe_2O_4nanoparticles were synthesized using thermal decomposition method. MnFe_2O_4nanoparticles were self-assembly with two block polymer of PEG-PCL to constructwater-soluble PEG-PCL-MnFe_2O_4nanomicelles. Endoglin specifically targeted polypeptideCL-1555was then bound to the surface of PEG-PCL-MnFe_2O_4nanomicelles,whosephysical properties were then studied. Using these nanomicelles, tumor angiogenesis andlymphangiogenesis were evaluated with MR imaging in vitro and in vivo, and the feasibilityof evaluating tumor angiogenesis and lymphangiogenesis in vivo were investigated in thisstudy.
Contents and Methods：
1. Synthesis and characterization of PEG-PCL-MnFe_2O_4nanomicelles
MnFe_2O_4nanoparticles were synthesized using thermal decomposition method.MnFe_2O_4nanoparticles were self-assembled with two block polymer of PEG-PCL toconstruct water-soluble PEG-PCL-MnFe_2O_4nanomicelles. The characteristics of theMnFe_2O_4nanoparticles and PEG-PCL-MnFe_2O_4nanomicelles were tested usingtransmission electron microscope (TEM), zeta-particle analyzer, infrared absorptionspectrum and inductively coupled plasma atomic emission spectrometry. The relaxation rateof MnFe_2O_4nanomicelles were measured with MR scanner.
2. Synthesis of CL-PEG-MnFe_2O_4nanomicelles and in vitro experiment
Endoglin specifically targeted polypeptide CL-1555was synthesized, which wasbound to the surface of PEG-PCL-MnFe_2O_4nanomicelles to constructed CL-PEG-MnFe_2O_4nanomicelles by crosslinking reaction. In order for the VECs and LECs to acquire thecharacteristics of breast cancer, VECs and LECs were incubated with breast cancer cells inMillicell cell culture. The incubated VECs and LECs were co-cultured with CL-PEG-MnFe_2O_4nanomicelles at molar iron concentration of0mmol/L、0.01mmol/L、0.02mmol/L、0.03mmol/L、0.04mmol/L、0.06mmol/L、0.08mmol/L、0.1mmol/L、0.2mmol/L、0.4mmol/L、0.6mmol/L and0.8mmol/L. The intracytoplasmic nanoparticleswere confirmed with Prussian blue iron staining,fluorescence microscopy and TEM. Non- incubated HDLECs and PEG-PCL-MnFe_2O_4nanomicelles were used as control. Labeledcells were suspended in PBS in EP tubes and axial MR imaging was performed on a3.0TMR scanner using an eight-channel phased-array head coil, with PBS as a control. Themorphologic features of cells in each concentration were observed to assess the toxicity ofCL-PEG-MnFe_2O_4nanomicelles on VECs. CCK-8assay kit was used to test the affect ofCL-PEG-MnFe_2O_4nanomicelles on cell proliferation activity. The labeled cells werepassaged, and the labeling efficiencies of labeled cells were observed. The contents ofmanganese and iron in passaged cells were measured using ICP-AES instrument, and theMR imaging were performed to observe the variation of MR signals.
3. Establishment of breast cancer xenograft and optimization of MR imaging
MR imaging were performed with small joints coil and dedicated small animal coil, andthe parameters for MR imaging were optimized to evaluate the practical value ofconventional MR scanner in nude mice. Superparamagnetic iron oxide (SPIO) nanoparticleswere intravenous through tail vein and retrobulbar vein. The value of retrobulbar veininjection of magnetic nanoparticles was evaluated using MR imaging.
4. MR molecular imaging of breast cancer xenograft based on CL-PEG-MnFe_2O_4nanomecelles
CL-PEG-MnFe_2O_4nanomicelles were intravenous administrated by retrobulbar vein.MR imaging was performed on a3.0T MR scanner using dedicated small animal coil. TheMR imaging sequences included spin echo (SE) T1-weighted imaging (T1WI), fast spinecho (FSE) T2-weighted imaging (T2WI), gradient echo (GRE) T2*-weighted imaging andFSE T2mapping with16echos, with PEG-PCL-MnFe_2O_4as a control. Rats were sacrificedafter MR imaging. Pathological and immunohistochemical analysis were performed in thearea consistent with the ROI of the MR images. Finally, the value of Endoglin-targeted MRimaging in detecting the angiogenesis and lymphangiogenesis in breast cancer wasevaluated by comparatively analyzing the MR images and pathological andimmunohistochemical results.
Results：
1. MnFe_2O_4nanoparticles appeared as round under TEM. The average size ofGoldMag particles was11nm with good monodisperse, zeta-particle size was11.18±1.72nm. The molar ratio of iron/manganese was2.13:1. After MnFe_2O_4nanoparticles were self-assembled with two block polymer of PEG-PCL, the size of PEG-PCL-MnFe_2O_4nanomicelles was enlarged, ranging from52nm to86nm, with a mean size of78.8±12.4nm. The absorption peak of the C=O around1710cm-1under the IR indicates the presenceof PEG. With increasing iron concentration, signal intensity (SI) decreasing after increasingfirst in SE T1WI, and decreased in FSE T2WI and GRE T2*WI. The signal intensity changesin FSE T2WI and GRE T2*WI were significantly stronger than that in SE T1WI, especiallyin GRE T2*WI (P<0.05).
2. The human umbilical vein endothelial cells (HUVECs) were isolated successfully.Incubated VECs were positive for CD105immunofluorescence staining, and the incubatedLECs showed red and green fluorescence after double staining with podoplanin and CD105.The fluorescence intensity of incubated VECs and LECs was significantly stronger than thatof non-incubated VECs and LECs. The cell labeling ratio with CL-PEG-MnFe_2O_4nanomicelles was significantly higher than that of PEG-PCL-MnFe_2O_4nanomicelles. Atiron concentrations of0ug/mL,0.5ug/mL,1ug/mL,2ug/mL,5ug/mL,10ug/mL,the labelingratios were, respectively,0%,26.95±4.38%,62.73±3.07%,82.34±3.67%,100%and100%forincubated HDLECs, and0%,15.61±3.42%,36.35±2.26%,52.31±3.23%,87.53±4.62%and100%for non-incubated HDLECs. Under the TEM, high electron density particles werefound in cytoplasm and lysosomes. SI gradually increased in SE T1WI, and decreased inFSE T2WI and GRE T2*WI. The signal intensity changes in GRE T2*WI were significantlystronger than that in FSE T2WI (P<0.05). The T2relaxation time decreased with increasingiron concentration. The SI of labeling cells with CL-PEG-MnFe_2O_4nanomicelles was lowerthan that with PEG-PCL-MnFe_2O_4nanomicelles, and the SI of incubated cell suspensionswas lower than that of non-incubated cells. The proliferative activity of VECs was affectedlittle when the iron concentration was lower than20ug/mL. When the iron concentrationreached50ug/mL, the proliferative activity of VECs was affected to some degree, and cellproliferation significantly affected when iron concentration reached100ug/mL, the activecells decreased significantly. As the cell passaging, labeling efficiency decreased. SIgradually increased in FSE T2WI. In SE T1WI, the SI of passage1(P1) cells was lower thanthat of PBS. However, the SI of P2and P3cells was higher than that of PBS. The P4cellsshowed isointense both in T1WI and T2WI. As the labeled cell passaging, the amount of irondecreased faster than manganese, and the molar ratio of iron/manganese gradually increased.
3. The establishment of breast cancer xenograft model by subcutaneous inoculationcell suspension was a simple operation. The tumor formation rate was100%, and the tumorincubation period was about1-2weeks. The characteristics of xenograft were similar to thatof human breast cancer. The signal to noise ratio (SNR), resolution and contrast withdedicated small animal coil were higher than that with small joints coil. The size ofxenograft in two weeks is about1cm3, and the SI of tumor is homogeneous. The tumordisplayed slightly hypointense in T1WI and hyperintense in T2WI images. The boundary ofthe tumor was clear, and no adhesion with adjacent structures. Necrosis occurred in largertumor (usually after3weeks), and the necrotic area showed hyperintense in T2WI images.The means of retrobulbar vein puncture is simple and easy. After injection of magneticnanoparticles by retrobulbar vein, nanoparticles accumulated in liver consistent with theclearance of nanoparticles in the plasma.
4. The enhancement degree at peripheral area of the tumor is higher than that in thecentral area after intravenous administration of CL-PEG-MnFe_2O_4nanomicelles. After60min clearance, the tumor displayed patchy or spot-like enhancement, which was mainlylimited to the peripheral areas of the tumors. However, the SI returned to the baseline after a60min recovery when the contrast agent was PEG-PCL-MnFe_2O_4nanomicelles. Under themicroscope, the blue-stained iron particles were observed around the cancer cell nest withPrussian blue iron staining. In the same area, blood vessels and lymphatics with brownstaining were observed, corresponding to immunohistochemical staining for CD105, CD34and podoplanin, respectively, indicating that the nanoparticles were combined with bloodvessels and lymphatic vessels of breast cancer. After intravenous injection ofCL-PEG-MnFe_2O_4nanomicelles, SI decreased immediately and the relative SI on T2WIweighted images were16%, thereafter, relative SI returned to about48%of the peak valueafter a60min recovery. However, the SI returned to the baseline after a30min recoverywhen the contrast was PEG-PCL-MnFe_2O_4nanomicelles.
Conclusion：
1. High quality MnFe_2O_4nanoparticles can be synthesized by thermal decomposition.The self-assembly PEG-PCL/MnFe_2O_4nanomicelles coated with amphiphilic blockcopolymer are more sensitive contrast agent for T2WI, which have stronger T2relaxivity than PEG-PCL/Fe3O4nanomicelles.
2. The separation method of HUVECs is convenient, simple, and can get many cells inone time. The HUVECs is a reliable cell for characterized study of endothelial cell. Themethod is a reliable way for gaining tumor-derived endothelial cells by co-culture ofendothelial cells and tumor cells. After sequence optimization, conventional clinical MRscanner could be used to image nude mice combining with a dedicated small animal coils.
3. Establishment of breast cancer xenograft by subcutaneous inoculation of cellsuspension in nude mice is simple, and the tumor formation rate is higher. The constructedbreast cancer is a reliable animal model for research of angiogenesis and lymphangio-genesis.
4. Venipuncture in the retrobulbar vein is more simple than the tail vein puncture,which is reliable route for intravenous administration of magnetic nanoparticles.
5. The CL-PEG-MnFe_2O_4nanomicelles combined with Endoglin targeting peptidescan specifically bind to tumor-derived VECs and LECs, which can bind to neovascularendothelial cells and lymphatic endothelial cells after intravenous injection, which could bedetected by MR imaging. The CL-PEG-MnFe_2O_4nanomicelles show no toxicity to cellswithin a certain concentration, which have potential application for target MR imaging oftumor neovascular and lymphatic vessels.
6. The paramagnetic manganese ions (Mn2+) can be released from MnFe_2O_4nanoparticles metabolized in cytoplasm, which can stay longer in the cell and could bedetected with T1WI imaging in a certain concentration. That is to say, the MnFe_2O_4nanoparticles are expected to apply for double-contrast agent of MR imaging.
血行转移和淋巴转移是恶性肿瘤常见的转移途径。自Folkman于1971年首次提出肿瘤发生、发展呈血管依赖性以来，肿瘤血管生成和抗肿瘤血管生成治疗一直是肿瘤研究的热点。近年来，随着一系列淋巴管内皮细胞（LECs）标记物的发现，对肿瘤淋巴管生成的研究也逐渐深入到了分子水平。许多肿瘤（尤其是上皮来源的肿瘤），如乳腺癌等，容易同时发生血行转移和淋巴转移。肿瘤微血管密度（MVD）和微淋巴管密度（LVD）与肿瘤的转移和预后密切相关。对于这些肿瘤，只检测其中某一条转移途径，往往达不到综合判断肿瘤是否易于发生远处转移的目的。同时，为了准确客观地评价肿瘤血管生成和淋巴管生成，还必须将肿瘤新生微血管和微淋巴管与正常组织的微血管和微淋巴管区分开来。
Endoglin又名CD105，是内皮细胞膜表达的糖蛋白，是转化生长因子-β（TGF-β）受体复合物的成分之一。它通过参与TGF-β受体的信号转导，调节内皮-间质的信号传递，参与血管生成，在再生组织、炎症及肿瘤组织的新生血管内皮细胞中过表达，并且Endoglin的表达水平与内皮细胞的增殖程度正相关；基于Endoglin染色的肿瘤MVD评估与肿瘤的预后密切相关。早期研究认为Endoglin仅在处于增殖状态的肿瘤组织血管内皮细胞（VECs）高表达，而在正常组织的VECs不表达或低表达，在LECs不表达。近来研究证明Endoglin也是LECs增殖的标记物，它在正常组织LECs不表达或低表达，而在肿瘤新生LECs高表达。因而，若将Endoglin作为分子靶标，应用于肿瘤的磁共振（MR）分子成像，则有望实现肿瘤微血管生成和微淋巴管生成的双重靶向成像。
目前，国内外均未见采用一种MR对比剂实现肿瘤微血管和微淋巴管特异性检测的相关报道。因此，本研究拟通过高温热解法合成超敏感MnFe_2O_4纳米粒，并用PEG-PCL两嵌段聚合物自组装形成高弛豫率的PEG-PCL-MnFe_2O_4纳米胶束，并进一步通过交联反应耦连能与Endoglin特异性结合的多肽CL-1555(氨基酸序列为AHKHVHHVPVRL)制备成CL-PEG-MnFe_2O_4分子探针，通过体外和体内实验证实该分子探针与肿瘤VECs和LECs的特异性结合，阐明靶向Endoglin分子探针介导的肿瘤微血管和微淋巴管MR成像作用，评价MR成像定量分析肿瘤新生血管及淋巴管的可能性和准确性。
研究内容及方法：
1. PEG-PCL-MnFe_2O_4纳米胶束的合成及表征高温热解法合成MnFe_2O_4纳米粒，利用部分末端功能化的PEG两亲性嵌段聚合物包裹MnFe_2O_4纳米粒自组装形成水溶液中单一分散的PEG-PCL-MnFe_2O_4纳米胶束。并采用透射电子显微镜（TEM）、zeta-粒度仪、红外吸收光谱(IR)及电感耦合等离子体原子发射光谱（ICP-AES）对纳米粒及纳米胶束的形态、粒径、分散性、包被物及铁锰构成比进行表征。用磁共振测量纳米胶束的弛豫率。
2. CL-PEG-MnFe_2O_4纳米胶束的构建及体外实验合成能与Endoglin特异性结合的短肽CL-1555，与PEG-PCL-MnFe_2O_4通过交联反应耦联制备靶向Endoglin的CL-PEG-MnFe_2O_4探针。将VECs及LECs与乳腺癌细胞共培养获取肿瘤源性VECs及LECs。以CL-PEG-MnFe_2O_4标记肿瘤源性VECs及LECs，普鲁士蓝染色及荧光显微镜观察探针与内皮细胞的结合情况，透射电子显微镜观察纳米粒进入胞浆的位置，以PEG-PCL-MnFe_2O_4胶束标记肿瘤源性VECs及LECs及CL-PEG-MnFe_2O_4标记人脐静脉内皮细胞（HUVECs）及LECs为对照。并对标记细胞进行MR成像，了解MR成像在检测标记细胞中的价值。CCK-8试剂盒检测CL-PEG-MnFe_2O_4对细胞增殖活性的影响。对标记后的细胞进行传代，观察传代后细胞标记的阳性率，测量传代细胞内锰、铁元素的量，并将每一代细胞进行MR扫描，观察随着细胞传代磁共振信号的变化规律。
3.裸鼠乳腺癌移植瘤模型的建立及MR成像优化采用小关节线圈及专用小动物线圈对裸鼠移植瘤模型进行MR成像，并对成像序列的参数进行优化，以期评价临床常规磁共振在小动物成像中的实用价值。通过尾静脉及球后静脉两种方式静脉注射超顺磁性氧化铁（SPIO）纳米粒，评价两种方法在裸鼠磁性纳米粒静脉给药中的可行性。
4. CL-PEG-MnFe_2O_4应用于裸鼠乳腺癌移植瘤模型的MR分子成像乳腺癌移植瘤模型经球后静脉注射CL-PEG-MnFe_2O_4探针，采用3.0T磁共振扫描仪进行SE T1WI、FSE T2WI、GRE T2*WI及T2mapping成像，以注射PEG-PCL-MnFe_2O_4为对照；扫描结束后，取肿瘤标本进行病理及免疫组化分析，并与MRI图像进行对照，了解CL-PEG-MnFe_2O_4探针对肿瘤新生血管及淋巴管的双重靶向效能。
结果：
1.所合成的MnFe_2O_4纳米粒呈圆形，粒径分布均匀，粒径在11nm左右，zeta-粒度仪结果显示纳米的大小为11.18±1.72nm，具有很好的单分散性。纳米粒的Fe/Mn摩尔浓度比为2.13:1。CL-PEG-MnFe_2O_4纳米胶束呈簇状及团状分布，由十几到几十个纳米粒聚集而成，胶束粒径为52-86nm， zeta平均粒径约78.8±12.4nm。IR显示在1710cm-1处出现一明显的羰基吸收峰。当铁摩尔浓度为：0mmol/L、0.01mmol/L、0.02mmol/L、0.03mmol/L、0.04mmol/L、0.06mmol/L、0.08mmol/L、0.1mmol/L、0.2mmol/L、0.4mmol/L、0.6mmol/L和0.8mmol/L时，随着浓度的增加，在SE T1WI，信号强度先升高后降低；在FSE T2WI，信号强度逐渐下降；在GRE T2*WI，信号强度随浓度的增加显著降低，GRE T2*WI较SE T1WI和FSE T2WI对纳米粒的检测更敏感（P＜0.05）。
2.成功分离脐静脉内皮细胞（HUVECs）。经诱导后的VECs和LECs免疫荧光染色显示，VECs呈CD105阳性，诱导后的LECs在podoplanin和CD105双染下呈绿色（podoplanin）和红色(CD105)荧光,诱导后的VECs和LECs荧光强度强于未诱导的内皮细胞。在相同铁浓度下，CL-PEG-MnFe_2O_4纳米胶束标记的肿瘤源性VECs和LECs的标记率较PEG-PCL-MnFe_2O_4高。在铁浓度为0μg/mL、0.5μg/mL、1μg/mL、2μg/mL、5μg/mL、10μg/mL时，诱导后和未诱导的LECs的标记率分别为0%、26.95±4.38%、62.73±3.07%、82.34±3.67%、100%、100%和0%、15.61±3.42%、36.35±2.26%、52.31±3.23%、87.53±4.62%、100%。在电子显微镜下标记细胞胞膜上、胞浆内及溶酶体可见包含高电子密度的内吞囊泡和细小颗粒；核周部分次级溶酶体破裂，周围胞浆内可见细小颗粒。在MR成像SE T1WI，信号强度呈缓慢升高；在FSE T2WI，信号强度逐渐下降；在GRE T2*WI，信号强度随浓度的增加显著降低。T2mapping显示随着浓度增加T2值逐渐降低，相对于PEG-PCL-MnFe_2O_4纳米胶束标记肿瘤源性VECs及LECs和CL-PEG-MnFe_2O_4纳米胶束标记VECs及LECs，CL-PEG-MnFe_2O_4纳米胶束标记的肿瘤源性VECs及LECs的T2值降低更明显，但随着铁浓度的增加，两者的差异逐渐减小。CCK-8细胞活性检测显示，在浓度小于20μg/mL时，纳米胶束对VECs细胞增殖活性影响不大，当浓度增加到50μg/mL时，细胞活性受到轻度影响，当铁浓度达到100μg/mL，细胞增殖活性受到明显影响，活性细胞数明显减少。标记细胞的普鲁士蓝染色显示，在铁浓度为50μg/mL以下时，细胞生长良好，细胞形态无明显改变，当铁浓度为100μg/mL时，活细胞数明显减少，细胞出现皱缩。随着标记细胞的传代，标记阳性率逐渐降低，在T2WI，磁共振信号逐渐升高；在T1WI，P1代呈低信号，P2、P3代呈稍高信号，P4代在T1WI、T2WI均呈等信号，ICP-AES显示随着细胞的传代，铁元素迅速减少，而锰元素减少较铁慢，Mn/Fe摩尔浓度比逐渐升高。
3.通过细胞悬液裸鼠皮下接种的方法建立乳腺癌动物模型操作简单、成瘤率为100%，肿瘤潜伏期约1-2周，所形成的肿瘤接近人体同类肿瘤本身的特性。采用小动物专用线圈所获得图像的信噪比、分辨率及对比度较关节小线圈高，图像信号显示更均匀。两周以内的移植瘤大小在1cm左右，肿瘤信号均匀，在T1WI呈稍低信号，T2WI呈较高信号，边界较清晰，可见包膜，与邻近结构无粘连；肿瘤生长三周过后，肿瘤内出现囊变、坏死，坏死区在T1WI呈低信号，T2WI呈高信号。球后静脉穿刺方法简单、易行，通过球后静脉注射SPIO后肝脏内纳米粒的蓄积符合纳米粒在血浆中的清除变化规律，球后静脉注射纳米粒肝脏的信号强度变化率较尾静脉注射大。
4.经球后静脉注射靶向探针CL-PEG-MnFe_2O_4后即刻及5min，肿瘤周围区增强程度高于中央区；注射CL-PEG-MnFe_2O_4后1h，肿瘤周边区仍呈点状或片状增强；而注射PEG-PCL-MnFe_2O_4后1h，肿瘤信号与增强前肿瘤信号基本相同。普鲁士蓝染色显示在肿瘤周边区可见大量蓝染颗粒，CD105、CD34及Podoplanin免疫组化染色显示相应区域局部血管及淋巴管丰富，与MRI图像肿瘤增强区相对应。靶向增强组在注射对比剂后即刻信号降低，最大信号强度变化率约为16%，之后肿瘤信号强度缓慢回升，1h后信号变化不明显，信号强度约为峰值信号强度的48%，时间信号强度曲线（TIC）呈下降-上升-平台型。对照组注射对比剂即刻，肿瘤信号强度降低，随时间延长，信号强度逐渐回升；30min时，信号强度接近注射前水平，此时，肿瘤信号与增强前无明显差异，TIC呈下降-上升型。
结论：
1.通过高温热解法能够合成高品质的MnFe_2O_4纳米粒。采用两亲嵌段聚合物自组装形成的PEG-PCL-MnFe_2O_4纳米胶束，具有较强的T2弛豫效能，在相同条件下较PEG-PCL-Fe3O4弛豫性能更高，是较敏感的T2WI对比剂。
2. HUVECs获取方便、分离方法简单、一次获取细胞量大，是研究内皮细胞特性可靠的细胞模型。通过内皮细胞与肿瘤细胞共培养能够使内皮细胞向肿瘤源性内皮细胞分化，是获取肿瘤源性内皮细胞的可靠方法。
3．细胞悬液裸鼠皮下接种的方法建立乳腺癌动物模型操作简单、成瘤率高，所构建的乳腺癌肿瘤是研究血管及淋巴管生成可靠的动物模型。
4.球后静脉穿刺较尾静脉穿刺更简单易行，是静脉注射磁性纳米粒可靠的给药途径。
5.耦连靶向Endoglin的结合肽后所构建的CL-PEG-MnFe_2O_4纳米胶束能够与肿瘤源性的VECs及LECs特异性结合，静脉注射后能够结合到乳腺癌新生血管及淋巴管内皮，并可以通过磁共振进行检测。在一定的浓度范围内对细胞无明显毒性，为应用MR特异性显示肿瘤新生血管和淋巴管提供了可能。
6. MnFe_2O_4纳米粒经细胞代谢后释放出顺磁性的锰离子（Mn2+），并能在细胞内较长时间停留，在一定浓度下可以通过T1WI成像进行检测，有望拓展MnFe_2O_4纳米粒在MR双对比领域的应用前景。
Background and Objective：
Hematogenous and lymphatic metastasis is the most common form of metastasis inpatients with malignant tumor. The angiogenesis and anti-angiogenesis therapy is alwaysthe hot point in cancer research since Folkman first proposed the hypothesis of tumordevelopment was dependent vascular in1971. In recent years, lymphangiogenesisgradually in depth study at the molecular level with a series of lymphatic endothelial cells(LECs) markers found on tumor. Many tumors (especially tumors of epithelial origin, suchas breast cancer, et al.) likely to simultaneously led to hematogenous and lymphaticmetastasis. Microvessel density (MVD) and lymphatic vessel density (LVD) are closelyrelated to the metastasis and prognosis of tumor. So, it is difficult to determine whether thetumor has occurred metastasis or not if we only focus on one metastatic pathway. At thesame time, In order to evaluate the angiogenesis and lymphangiogenesis accurately, wemust differentiate the tumor neoangiogenesis and neonatal lymphatic vessels from themicrovessels in the normal tissue.
Endoglin, also known as CD105, is a glycoprotein expressed in endothelial cellmembrane, is one of the transforming growth factor-β (TGF-β) receptor complex. Endoglinregulate the signal transduction between endothelial cell and interstitial tissue through theTGF-β, and promote the angiogenesis. Endoglin overexpressed in neonatal vascularendothelial cells in regenerated tissue, inflammation and tumor tissue. The expression levelsand degree of Endoglin is closely related to the endothelial cell proliferation. The MVDbased on Endoglin staining is closely related to the tumor prognosis. Endoglin is also theproliferation marker of LECs, which is overexpressed in neonatal lymphatic vessel, but notexpressed in the LECs of normal tissue. Thus, it is expected to achieve dual targeting imaging of breast cancer angiogenesis and lymphangiogenesis if Endoglin was used as amolecular target in magnetic resonance (MR) molecular imaging.
To our knowledge, there is no relevant report on simultaneous detection of tumormicrovessel and microlymphatic vessel using a MR contrast agent. In this study, MnFe_2O_4nanoparticles were synthesized using thermal decomposition method. MnFe_2O_4nanoparticles were self-assembly with two block polymer of PEG-PCL to constructwater-soluble PEG-PCL-MnFe_2O_4nanomicelles. Endoglin specifically targeted polypeptideCL-1555was then bound to the surface of PEG-PCL-MnFe_2O_4nanomicelles,whosephysical properties were then studied. Using these nanomicelles, tumor angiogenesis andlymphangiogenesis were evaluated with MR imaging in vitro and in vivo, and the feasibilityof evaluating tumor angiogenesis and lymphangiogenesis in vivo were investigated in thisstudy.
Contents and Methods：
1. Synthesis and characterization of PEG-PCL-MnFe_2O_4nanomicelles
MnFe_2O_4nanoparticles were synthesized using thermal decomposition method.MnFe_2O_4nanoparticles were self-assembled with two block polymer of PEG-PCL toconstruct water-soluble PEG-PCL-MnFe_2O_4nanomicelles. The characteristics of theMnFe_2O_4nanoparticles and PEG-PCL-MnFe_2O_4nanomicelles were tested usingtransmission electron microscope (TEM), zeta-particle analyzer, infrared absorptionspectrum and inductively coupled plasma atomic emission spectrometry. The relaxation rateof MnFe_2O_4nanomicelles were measured with MR scanner.
2. Synthesis of CL-PEG-MnFe_2O_4nanomicelles and in vitro experiment
Endoglin specifically targeted polypeptide CL-1555was synthesized, which wasbound to the surface of PEG-PCL-MnFe_2O_4nanomicelles to constructed CL-PEG-MnFe_2O_4nanomicelles by crosslinking reaction. In order for the VECs and LECs to acquire thecharacteristics of breast cancer, VECs and LECs were incubated with breast cancer cells inMillicell cell culture. The incubated VECs and LECs were co-cultured with CL-PEG-MnFe_2O_4nanomicelles at molar iron concentration of0mmol/L、0.01mmol/L、0.02mmol/L、0.03mmol/L、0.04mmol/L、0.06mmol/L、0.08mmol/L、0.1mmol/L、0.2mmol/L、0.4mmol/L、0.6mmol/L and0.8mmol/L. The intracytoplasmic nanoparticleswere confirmed with Prussian blue iron staining,fluorescence microscopy and TEM. Non- incubated HDLECs and PEG-PCL-MnFe_2O_4nanomicelles were used as control. Labeledcells were suspended in PBS in EP tubes and axial MR imaging was performed on a3.0TMR scanner using an eight-channel phased-array head coil, with PBS as a control. Themorphologic features of cells in each concentration were observed to assess the toxicity ofCL-PEG-MnFe_2O_4nanomicelles on VECs. CCK-8assay kit was used to test the affect ofCL-PEG-MnFe_2O_4nanomicelles on cell proliferation activity. The labeled cells werepassaged, and the labeling efficiencies of labeled cells were observed. The contents ofmanganese and iron in passaged cells were measured using ICP-AES instrument, and theMR imaging were performed to observe the variation of MR signals.
3. Establishment of breast cancer xenograft and optimization of MR imaging
MR imaging were performed with small joints coil and dedicated small animal coil, andthe parameters for MR imaging were optimized to evaluate the practical value ofconventional MR scanner in nude mice. Superparamagnetic iron oxide (SPIO) nanoparticleswere intravenous through tail vein and retrobulbar vein. The value of retrobulbar veininjection of magnetic nanoparticles was evaluated using MR imaging.
4. MR molecular imaging of breast cancer xenograft based on CL-PEG-MnFe_2O_4nanomecelles
CL-PEG-MnFe_2O_4nanomicelles were intravenous administrated by retrobulbar vein.MR imaging was performed on a3.0T MR scanner using dedicated small animal coil. TheMR imaging sequences included spin echo (SE) T1-weighted imaging (T1WI), fast spinecho (FSE) T2-weighted imaging (T2WI), gradient echo (GRE) T2*-weighted imaging andFSE T2mapping with16echos, with PEG-PCL-MnFe_2O_4as a control. Rats were sacrificedafter MR imaging. Pathological and immunohistochemical analysis were performed in thearea consistent with the ROI of the MR images. Finally, the value of Endoglin-targeted MRimaging in detecting the angiogenesis and lymphangiogenesis in breast cancer wasevaluated by comparatively analyzing the MR images and pathological andimmunohistochemical results.
Results：
1. MnFe_2O_4nanoparticles appeared as round under TEM. The average size ofGoldMag particles was11nm with good monodisperse, zeta-particle size was11.18±1.72nm. The molar ratio of iron/manganese was2.13:1. After MnFe_2O_4nanoparticles were self-assembled with two block polymer of PEG-PCL, the size of PEG-PCL-MnFe_2O_4nanomicelles was enlarged, ranging from52nm to86nm, with a mean size of78.8±12.4nm. The absorption peak of the C=O around1710cm-1under the IR indicates the presenceof PEG. With increasing iron concentration, signal intensity (SI) decreasing after increasingfirst in SE T1WI, and decreased in FSE T2WI and GRE T2*WI. The signal intensity changesin FSE T2WI and GRE T2*WI were significantly stronger than that in SE T1WI, especiallyin GRE T2*WI (P<0.05).
2. The human umbilical vein endothelial cells (HUVECs) were isolated successfully.Incubated VECs were positive for CD105immunofluorescence staining, and the incubatedLECs showed red and green fluorescence after double staining with podoplanin and CD105.The fluorescence intensity of incubated VECs and LECs was significantly stronger than thatof non-incubated VECs and LECs. The cell labeling ratio with CL-PEG-MnFe_2O_4nanomicelles was significantly higher than that of PEG-PCL-MnFe_2O_4nanomicelles. Atiron concentrations of0ug/mL,0.5ug/mL,1ug/mL,2ug/mL,5ug/mL,10ug/mL,the labelingratios were, respectively,0%,26.95±4.38%,62.73±3.07%,82.34±3.67%,100%and100%forincubated HDLECs, and0%,15.61±3.42%,36.35±2.26%,52.31±3.23%,87.53±4.62%and100%for non-incubated HDLECs. Under the TEM, high electron density particles werefound in cytoplasm and lysosomes. SI gradually increased in SE T1WI, and decreased inFSE T2WI and GRE T2*WI. The signal intensity changes in GRE T2*WI were significantlystronger than that in FSE T2WI (P<0.05). The T2relaxation time decreased with increasingiron concentration. The SI of labeling cells with CL-PEG-MnFe_2O_4nanomicelles was lowerthan that with PEG-PCL-MnFe_2O_4nanomicelles, and the SI of incubated cell suspensionswas lower than that of non-incubated cells. The proliferative activity of VECs was affectedlittle when the iron concentration was lower than20ug/mL. When the iron concentrationreached50ug/mL, the proliferative activity of VECs was affected to some degree, and cellproliferation significantly affected when iron concentration reached100ug/mL, the activecells decreased significantly. As the cell passaging, labeling efficiency decreased. SIgradually increased in FSE T2WI. In SE T1WI, the SI of passage1(P1) cells was lower thanthat of PBS. However, the SI of P2and P3cells was higher than that of PBS. The P4cellsshowed isointense both in T1WI and T2WI. As the labeled cell passaging, the amount of irondecreased faster than manganese, and the molar ratio of iron/manganese gradually increased.
3. The establishment of breast cancer xenograft model by subcutaneous inoculationcell suspension was a simple operation. The tumor formation rate was100%, and the tumorincubation period was about1-2weeks. The characteristics of xenograft were similar to thatof human breast cancer. The signal to noise ratio (SNR), resolution and contrast withdedicated small animal coil were higher than that with small joints coil. The size ofxenograft in two weeks is about1cm3, and the SI of tumor is homogeneous. The tumordisplayed slightly hypointense in T1WI and hyperintense in T2WI images. The boundary ofthe tumor was clear, and no adhesion with adjacent structures. Necrosis occurred in largertumor (usually after3weeks), and the necrotic area showed hyperintense in T2WI images.The means of retrobulbar vein puncture is simple and easy. After injection of magneticnanoparticles by retrobulbar vein, nanoparticles accumulated in liver consistent with theclearance of nanoparticles in the plasma.
4. The enhancement degree at peripheral area of the tumor is higher than that in thecentral area after intravenous administration of CL-PEG-MnFe_2O_4nanomicelles. After60min clearance, the tumor displayed patchy or spot-like enhancement, which was mainlylimited to the peripheral areas of the tumors. However, the SI returned to the baseline after a60min recovery when the contrast agent was PEG-PCL-MnFe_2O_4nanomicelles. Under themicroscope, the blue-stained iron particles were observed around the cancer cell nest withPrussian blue iron staining. In the same area, blood vessels and lymphatics with brownstaining were observed, corresponding to immunohistochemical staining for CD105, CD34and podoplanin, respectively, indicating that the nanoparticles were combined with bloodvessels and lymphatic vessels of breast cancer. After intravenous injection ofCL-PEG-MnFe_2O_4nanomicelles, SI decreased immediately and the relative SI on T2WIweighted images were16%, thereafter, relative SI returned to about48%of the peak valueafter a60min recovery. However, the SI returned to the baseline after a30min recoverywhen the contrast was PEG-PCL-MnFe_2O_4nanomicelles.
Conclusion：
1. High quality MnFe_2O_4nanoparticles can be synthesized by thermal decomposition.The self-assembly PEG-PCL/MnFe_2O_4nanomicelles coated with amphiphilic blockcopolymer are more sensitive contrast agent for T2WI, which have stronger T2relaxivity than PEG-PCL/Fe3O4nanomicelles.
2. The separation method of HUVECs is convenient, simple, and can get many cells inone time. The HUVECs is a reliable cell for characterized study of endothelial cell. Themethod is a reliable way for gaining tumor-derived endothelial cells by co-culture ofendothelial cells and tumor cells. After sequence optimization, conventional clinical MRscanner could be used to image nude mice combining with a dedicated small animal coils.
3. Establishment of breast cancer xenograft by subcutaneous inoculation of cellsuspension in nude mice is simple, and the tumor formation rate is higher. The constructedbreast cancer is a reliable animal model for research of angiogenesis and lymphangio-genesis.
4. Venipuncture in the retrobulbar vein is more simple than the tail vein puncture,which is reliable route for intravenous administration of magnetic nanoparticles.
5. The CL-PEG-MnFe_2O_4nanomicelles combined with Endoglin targeting peptidescan specifically bind to tumor-derived VECs and LECs, which can bind to neovascularendothelial cells and lymphatic endothelial cells after intravenous injection, which could bedetected by MR imaging. The CL-PEG-MnFe_2O_4nanomicelles show no toxicity to cellswithin a certain concentration, which have potential application for target MR imaging oftumor neovascular and lymphatic vessels.
6. The paramagnetic manganese ions (Mn2+) can be released from MnFe_2O_4nanoparticles metabolized in cytoplasm, which can stay longer in the cell and could bedetected with T1WI imaging in a certain concentration. That is to say, the MnFe_2O_4nanoparticles are expected to apply for double-contrast agent of MR imaging.
引文
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