L-选择素配体靶向性USPIO-PBCA纳米微粒的制备及其在淋巴结的MRI应用
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摘要
研究背景和目的
一、研究背景：
淋巴结是哺乳动物重要的免疫器官,淋巴通路是恶性肿瘤转移的重要途径,临床上判断淋巴结是否累及,是肿瘤分期、治疗方案选择、疾病预后判断的重要参考指标。淋巴结活检具有较大价值,但是进行淋巴结深部活检或临床治疗后复查常常比较困难,寻找一种简单、安全、无创的淋巴结检查方法受到广泛的重视。
传统上,判断淋巴结的转移与否常采用测定淋巴结径线的方法,此方法作为常规计算机断层扫描(Computed Tomography, CT)或磁共振成像(Magnetic Resonance Imaging, MRI)对淋巴结的定性诊断价值仍有很大的争议。超声波检查淋巴结血管的变化在鉴别淋巴结的良恶性病变中具有一定的意义,但良恶性淋巴结的征象有较多的重叠,超声的诊断准确率偏低。X线淋巴结造影术方法复杂、费时,有一定的并发症,不能满意显示淋巴结细节等特点。正电子发射计算机体层扫描(Positron Emission Tomography, PET)/CT从分子代谢的角度判断淋巴结的转移情况,但分辨率低,且价格昂贵。
采用对比剂增强的MR淋巴管成像是一种新型无创性的检测方法,目前使用最广泛、较理想的对比剂是超顺磁性氧化铁(Superparamagnetic Iron Oxide, SPIO)类对比剂,这是一种网状内皮系统被动靶向对比剂,能够在T2加权成像(T2-weighted Imaging, T2WT)序列上产生负性增强的作用。直径<30nm的SPIO称为超小超顺磁性氧化铁(Ultrasmall Superparamagnetic Iron Oxide, USPIO),它的MRI增强扫描可以发现位于淋巴结内的局限性转移灶,而与淋巴结是否增大无关。但是USPIO是被淋巴结髓质的吞噬细胞吞噬而显像的,属于被动靶向方式,存在不少问题,可能会有假阴性、假阳性的发生,影响诊断的正确性。
L-选择素(L-selectin)存在于正常的淋巴细胞表面上,其正常功能是作为淋巴细胞到达外周淋巴结归巢的受体。淋巴结的高内皮微静脉(High Endothelial Venules, HEV)内皮细胞上、迷路及髓质中,均有L-选择素配体的高度表达,L-选择素配体的核心抗原是MECA-79,其单抗具有与L-选择素相似的作用,能高度特异性与L-选择素配体高表达区结合,已有文献表明抗MECA-79单抗修饰的超声对比剂可以主动靶向正常淋巴结；聚氰基丙烯酸正丁酯(Polybutylcyanoacrylate, PBCA)以安全无毒、生物相容性好等特点成为目前最受重视的药物载体,那么PBCA经此抗体修饰以后,以它为载体携带超顺磁性物质(比如USPIO)可以制成MR对比剂,这种MR增强扫描应该可以实现主动靶向淋巴结,据我们己知的文献尚未见类似报道。
二、研究目的：
1、在弱酸性的条件下,将USPIO颗粒包被在PBCA纳米球内部,碱性条件下处理之后自动生成羧基,USPIO-PBCA经交联剂与链霉亲和素反应,再与生物素化MECA-79单抗,通过生物素-亲和素系统修饰到USPIO-PBCA纳米微粒外壳上,从而制备L-选择素配体靶向性的USPIO-PBCA纳米微粒；
2、动物体外实验,将生物素化MECA-79单抗在正常兔的新鲜淋巴结上做免疫组化实验检测该抗体的特异性靶向能力；
3、正常新西兰大白兔活体动物实验,将制备出的抗MECA-79-USPIO-PBCA纳米微粒通过足背组织间隙注射的方式做MRI增强扫描,在不同时间点检测对正常兔胭窝淋巴结的定性强化效果,病理检查证实。
方法
一、制备与表征
1、USPIO-PBCA纳米微粒的制备及检测
将氰基丙烯酸正丁酯(Butylcyanoacrylate, BCA)单体用CH2-Cl2稀释(VCH2Cl2:VBCA=3:1),配备酸性条件(pH6.4)的缓冲溶液(KH2PO4-NaOH)50ml,在溶液中融入浓度约为5%非离子表面活性剂Dextran-70,将浓度为11.2mg/ml的USPIO滴加入混合溶液中,搅拌5min,搅拌速率为800rpm。加大搅拌速率至1200rpm,缓慢逐滴滴加1.2%BCA单体入混合液中,持续乳化聚合5h,终止反应后减压抽滤,即得PBCA磁性纳米球稳定混悬液。随后用0.45μm微孔滤膜过滤,将滤液存于4℃冰箱内备用。
Malvern-3000HS激光粒度分析仪测纳米微粒的粒径和分布：取上述USPIO-PBCA混合液透析48h,除去游离的USPIO、Dextran-70,取透析过的USPIO-PBCA溶液用Malvern-3000HS激光粒度分析仪测定粒径及其分布。
透射电镜观察纳米微粒的形态：取少量上述经透析过USPIO-PBCA,在透射电镜下观察USPIO-PBCA大体形态。
振动样品磁强计检测微粒的磁学特性：在室温下,将约0.5mg冻干粉状USPIO-PBCA纳米微粒装入直径1cm的试管,密封后置入振动样品磁强计,逐渐加大磁场强度,并从显示屏观察磁化曲线,直到样品的磁化强度不再增加为止,记录磁化曲线。
磁共振扫描测T2时间及T2弛豫率：取USPIO-PBCA溶液适量,分别加水稀释至铁浓度为0.04、0.06、0.08、0.10、0.12、0.14、0.16mmol/L,在MR机器(3.0T,美国GE公司,Signa EXCIT HD)上扫描,采用8通道头部线圈,层厚3.0mm,间隔0.5mm,扫描序列采用T2map:重复时间(Time of Repetiton,TR)3000ms,回波时间(Time of Echo,TE)20ms、40ms、60ms、80ms,均做冠状位扫描。测定T2值：将原始数据导入ADW4.3工作站,测量USPIO-PBCA的T2弛豫时间值。
计算USPIO-PBCA T2弛豫率：T2弛豫率定义为含铁浓度与1/T2直线方程的斜率。
2、链霉亲和素连接USPIO-PBCA微粒及检测
将获得的初始USPIO-PBCA在碱性条件下(pH=9)作用一个小时,使其表面产生羧基基团；用灭菌磷酸盐缓冲液(Phosphate Buffered Saline, PBS)将USPIO-PBCA稀释至含铁量为0.52μM/ml,,将含1-乙基-3-(3-二甲氨基丙基)-碳二亚胺盐酸盐(1-ehy1-3-(3-dimethyl aminopropyl)carbodiimide hydrochloride, EDC)的PBS溶液加入其中,调整溶液pH至7.0,然后加入链霉亲和素的PBS溶液,4℃反应过夜,得到链霉亲和素标记的USPIO-PBCA纳米微粒。反应摩尔比：USPIO-PBCA:链霉亲和素：EDC为1：10：25(EDC浓度在0.15g/L最佳)。
用生物素化的生物素-异硫氰酸荧光黄(fluorescein isothiocyanate, FITC)与链霉亲和素化的USPIO-PBCA纳米微粒反应,用荧光显微镜观察荧光程度来评价链霉亲和素是否成功接到USPIO-PBCA纳米微粒外壳上
用滴定法将生物素化的FITC逐滴滴加在链霉亲和素化的USPIO-PBCA溶液里,采用流式细胞仪观察荧光峰的移动情况。
3、链霉亲和素标记的USPIO-PBCA与生物素化MECA-79单抗的链接加入5μg的对应生物素化的MECA-79单抗在含有107链霉亲和素标记的USPIO-PBCA的混悬液中,孵育10min,即可得抗MECA-79-USPIO-PBCA纳米微粒。
4、MECA-79单抗标记的USPIO-PBCA微粒理化特性的测定
投射电子显微镜观察已制备纳米微粒的表观形态特征,粒度测定测试MECA-79单抗标记的USPIO-PBCA纳米微粒的分布、粒径。
二、MECA-79单抗在正常兔新鲜淋巴结的靶向性实验
实验分组方法：
1)、实验组：生物素化的MECA-79单抗；
2)、对照组：另外一种生物素化的同源性IgM。
分别作免疫组化,观察抗体的靶向性情况。
选用正常的新西兰大白兔3只,麻醉后每只动物取双侧胭窝淋巴结各1枚,一共6枚,预冷的生理盐水反复漂洗3-5次,漂净血水,吸水滤纸吸干水分,将新鲜的正常淋巴结立刻放入-70℃快速冷冻,丙酮固定,行冰冻切片,片厚8μm,非特异的结合点先用5%(0.1mol/L PBS配制)山羊血清白蛋白进行封闭(37℃,孵育.1小时,立即用PBS漂洗过夜,4℃)；室温下,切片放在含1%生物素化单抗的山羊血清白蛋白(1：100)孵育1小时,再用3%的H2O2(0.1mol/LPBS配制)处理30min,以封闭内源性的辣根过氧化物酶的活性；然后O.1mol/LPBS漂洗3x5min,再滴加一种外源性的亲和素性过氧化物酶孵育1h,显微镜下观察显色情况。
三、MECA-79单抗标记的USPIO-PBCA纳米微粒对正常兔淋巴结的靶向性成像研究
1、实验分组：将18只正常新西兰大白兔随机分为三组,每组6只。
1)靶向组：正常新西兰大白兔,经一侧足趾间隙注射含Fe15μmol的抗MECA-79-USPIO-PBCA对比剂；
2)对照组1：正常新西兰大白兔,经一侧足趾间隙注射含Fe15μmol的USPIO-PBCA对比剂；
3)对照组2：正常新西兰大白兔,经一侧足趾间隙注射含Fe15μmol的USPIO对比剂。
2、实验步骤：
1)麻醉：3%戊巴比妥钠(20mg/Kg)经耳缘静脉给药,酌情添加少量安定,麻醉动物。
2)固定：以仰卧位的方式将动物固定在一合适木板上,左右尽量对称。
3、扫描设备及参数
1)扫描设备：GE Signa Excite3.0T MR磁共振成像系统,采用膝关节线圈。
2)扫描序列及参数：冠状位、矢状位T2WI(TR/TE:5000/85ms,回波链(Echo Train Length, ETL):16,层厚：2mm,视野(Field of View, FOV):12cm,扫描矩阵：256×256,重复次数：3)；冠状位、矢状位T2WI,2D梯度回波T2*加权成像(T2*-Weighted Imaging,T2*WI)(TR/TE:400/24ms,反转角：20-,层厚：2mm, FOV:12cm,扫描矩阵：256×256,脉冲重复激发次数(Number of Excitations, NEX):2);矢状位、横断位T1加权成像(T1-Weighted Imaging,T1WI)自旋回波序列(TR/TE:400/12ms,层厚：2mm, FOV:12cm,扫描矩阵：256×256, NEX：2),以上序列需要平扫及增强扫描。
3)对比剂增强扫描方式：向兔的一侧足趾间隙局部注射对比剂(一侧肢体注射总容量0.2m1),注射后1,2,8,12,24,48h分别成像。MR扫描完成后,将数据传至后处理工作站,进行后续的MRI图像分析。
4、数据处理
分别测定注射对比剂侧的淋巴结信号强度、肌肉组织信号强度,在背景区域选取较大的ROI,测背景噪声的标准差。计算各组淋巴结的信噪比。
5、统计学分析
采用SPSS13.0软件包,采用重复测量的方差分析(统计方法：Bonferroni)对比增强前后各组淋巴结及肌肉组织信噪比(Signal to Noise Ratio, SNR)是否有显著性差异。用单因素方差分析(统计方法：Bonferroni)分析组间不同时间点淋巴结及肌肉组织SNR是否有显著性差异,P<0.05认为有显著性差异。
6、病理学检查
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,取出胭窝淋巴结,然后用10-15%中性福尔马林浸泡、固定,常规石蜡包埋、切片,片厚1.5μm,行普鲁士蓝染色(Prussion Blue Staining),分别观察实验组、对照组1和对照组2淋巴结组织常规光镜下的形态及组织内含Fe的情况,与影像学表现进行对照分析。
结果
一、用Malvern-3000HS激光粒度仪测定的USPIO-PBCA平均粒径约为159nm,多分散系数为0.23。
二、透射电镜下观察USPIO-PBCA纳米微粒呈球形、类球形,分布较均匀,表面光滑、平整,粒子之间无粘连,粒子平均粒径大小约为60-70nm,提示制备出了小粒径、高稳定性的USPIO-PBCA纳米颗粒。
三、振动样品磁强计磁学特性测试结果显示制备出的USPIO-PBCA纳米颗粒具有超顺磁性。
四、经磁共振T2弛豫时间扫描,随着铁浓度的增加,USPIO-PBCA的T2值逐渐下降,所得样品的T2弛豫率0.156x106mol-1.s-1。
五、荧光显微镜和流式细胞仪检测结果,均提示USPIO-PBCA已经成功链霉亲和素化,并且具备与生物素化的单抗反应活性。
六、扫描电子显微镜观察已制备纳米微粒的表观形态特征,显示纳米微粒成球规整,呈圆形或者椭圆形,分布均匀,彼此不粘连,粒径约80nm。粒度测定显示粒度分布均匀,粒径约220.3nm,分散系数为0.318。
七、正常兔新鲜淋巴结辣根过氧化物酶免疫组化结果显示,靶向组MECA-79单抗对正常淋巴结有特异性染色,对照组普通IgM单抗则没有特异性染色。
八、磁共振扫描正常兔胭窝淋巴结和SPSS统计学分析结果显示,在T2*WI、T2WI序列上,靶向实验组的淋巴结SNR降低低于对照组1和对照组2淋巴结,差异有统计学意义,同一组间不同时间点差异也有统计学意义,不同组间和不同时间点有交互效应；在T1WI序列上,三组淋巴结的SNR差异有统计学意义,不同时间点差异也有统计学意义,不同组间和时间点无交互效应；三组肌肉组织SNR在T2*WI、T2WI、T1WI序列上,不同组间及组内不同时间点差异无统计学意义。
九、病理检查普鲁士蓝染色结果显示,靶向组注射侧的兔淋巴结内含有较多蓝染的铁颗粒,分布范围较广,对照组两组内蓝染的铁颗粒相对较少,且主要局限于淋巴结的髓质内。
结论
一、在弱酸性条件下,成功制备出具有超顺磁性的抗MECA-79-USPIO-P BCA靶向性纳米微粒,粒子大小较均匀,表面光滑、平整,为后续动物实验打下基础。
二、MECA-79单抗可以特异性靶向兔的正常淋巴结的副皮质区,可以间接说明制备出的抗MECA-79-USPIO-PBCA纳米微粒可以特异性靶向兔正常淋巴结。
三、动物活体实验表明,抗MECA-79-USPIO-PBCA纳米微粒对正常淋巴结具有特异靶向性,可以使正常淋巴结在T2*WI、T2WI、T1WI上SNR降低；而且对照实验表明靶向组的负性强化作用要明显强于两组对照组,普鲁士蓝染色结果显示靶向组的淋巴结内蓝染的铁颗粒较多,而且范围更广,说明制备出的抗MECA-79-USPIO-PBCA纳米微粒不仅能够被单核-吞噬系统所吞噬,而且能够主动靶向淋巴结的副皮质区。
Background and Objective
Background
Lymph nodes are important immune organs in mammalian and the lymphatic pathway is an important route of tumor metastasis. To judge the lymph nodes involved or not, it is a major index for tumor stage, treatment alternatives, disease prognosis. Although with great values, it has many difficulties in lymph nodes biopsies in deep tissue or review after clinical treatment. Nowadays, to find a simple, safe, non-invasive lymph node examination method has been received wide attentions.
Traditionally, to judge lymph node metastasis or not, it was often to use the method of determination of lymph node size. But, in the qualitative diagnostic value of lymph node, this method still has considerable controversy in routine Computed Tomography (CT) or Magnetic Resonance Imaging (MRI) scan. Ultrasound examing the vascular change of lymph node has some significance in the identification of the benign or malignant lesions in lymph nodes. Because of the overlap signs between in the benign and the malignant lymph nodes, the diagnostic accuracy of ultrasound is ralertively low. X-ray lymphadenography are complex, time-consuming, with some complications and can not satisfactorily display the details of lymph nodes. PET (Positron Emission Tomography)/CT could determine the lymph node metastasis from the perspective of molecular metabolism, but the resolution is low.
Contrast-enhanced MR lymphatic imaging is a new noninvasive detection method. Currently the Superparamagnetic Iron Oxides (SPIO)s contrast agents are the most widely used and the preferred ones which are a kind of passively targeting to reticuloendothelial system contrast agents with negative enhancement effect on the T2WI sequences. Ultrasmall Superparamagnetic Iron Oxides (USPIO)(the diameter less than30nm in SPIO) enhancement MRI scans can find localized metastases in the lymph nodes independent of the sizes of lymph nodes. But USPIO particles were engalfed by the phagocytic cells in the lymph node medulla to image. This enhancement MRI is a passive targeting method with many problems, which may cause false positives and false negtives and affect the accuracy of diagnosis.
L-selectins exit in the surface of normal lymphocytes, as the homing receptors which lead peripheral lymphocytes to reach lymph nodes. L-selectin ligands highly express in the endothelial cells of high endothelial veins, labyrinths and medullas of lymph nodes and its core antigens are MECA-79monoclonal antibodies, which have a similar role with L-selectin and highly specific to combine with the areas where L-selectin ligand highly expressed in. Literature suggested that ultrasound contrast agent modified by MECA-79monoclonal antibody could target initiatively to normal lymph nodes. Therefore, superparamagnetic substances (for example USPIO) carried by the antibody may target initiatively to lymph nodes in MR enhancement scan. Being of safe and good biocompatibility characteristics, Polybutylcyanoacrylate (PBCA) was the most important drug carrier. So, after PBCA were modified by this antibody, these nanoparticles which carryed superparamagneticsubstances (such as USPIO) could be prepared as MR contrast agent, this enhanced MR scan should be able to target to the lymph nodes actively. As we known that no similar literatures have reported.
Objectives of Study
1、In the weakly acidic conditions, the USPIO particles were coated in the PBCA nanospheres. After processing under alkaline conditions to generate carboxyls automatically, USPIO-PBCA particles reacted with streptavidins by crosslinking agent. Then, biotinylated MECA-79monoclonal antibodies were modified to USPIO-PBCA nanoparticles shells by biotin-avidin system, which made nanoparticles initiativly targeting to L-selectin ligands of lymph nodes.
2、The animal in vitro experiments:the specific targeting abilities of the biotinylated MECA-79monoclonal antibodies were detected by immunohistochemistry in fresh lymph nodes of normal rabbits.
3、Normal live New Zealand white rabbits experiments:by injection of anti-MECA-79-of USPIO-PBCA nanoparticles through the dorsal gap injection, the qualitative enhancement on normal rabbit popliteal lymph nodes at different time points by MRI scan, then by confirmed in pathology.
Methods
I Preparation and characterization
1、Synthesis and detection of USPIO-PBCA nanoparticles
BCA monomer was diluted by CH2-Cl2(VCH2ci2:VBCA=3:1).The non-ionic surfactant of5%Dextran-70was dissolved into the acidic (pH6.4) buffer solution (KH2PO4-NaOH) solution and the concentration of11.2mg/ml USPIO was dropped to the mixed solution of50ml. Afer stirring5minutes with the stirring speed of800rpm to1200rpm incresingly,1.2%concentration of the butylcyanoacrylate (BCA) monomer was slowly added into the mixture solution drop by drop. After emulsion polymerization continuously for5hours, the reaction was terminated. After this solution was filtered by vacuum pumb, the stable suspension of PBCA magnetic nanoparticles was prepared. Following filtred by0.45μm microporous filtration membrane, the filtrate was kept in the refrigerator of4℃for use.
Size and distribution of particle nanoparticles:Malvern-3000HS laser particle size analyzer: Take the above of USPIO-PBCA mixture to dialysis for48hours to remove of free USPIO and Dextran-70. The dialysised USPIO-PBCA solution was detected about the size and distribution by Malvern-3000HS laser particle size analyzer.
The morphology of nanoparticles by transmission electron microscopy:Take a small amount of the above of dialysised USPIO-PBCA, then the gross shape of it was observed by transmission electron microscopy.
The measured T2relaxation time and the calculated T2relaxation rate by Magnetic resonance scan:appropriate amount of USPIO-PBCA solution was diluted by water to the iron concentration of0.04,0.06,0.08,0.10,0.12,0.14,0.16mmol/L respectively, then scanned in the MR machine (3.0T U.S. company GE, Sigma EXCIT HD) with an8-channel head coil, thickness of3.0mm, interval of0.5mm, the scanning sequence T2map:time of repetion (TR) of3000ms, time of echo(TE) of20ms,40ms,60ms,80ms, all with a coronal scan. Determination of T? values: raw data were imported to ADW4.3workstation and USPIO-PBCA T2relaxation time values were measured.
Calculate the T2relaxation rate of USPIO-PBCA:T2relaxation rate is defined as the slope of the linear equation about the iron concentration and1/T2.
2、Preparation and detection Streptavidin-linked USPIO-PBCA particles
The prepared USPIO-PBCA particles were dealed with under alkaline conditions (pH=9) for an hour, so carboxyl groups generated on the surfaces of particles. Thses USPIO-PBCA particles were diluted to the iron concentration of USPIO-PBCA0.52μM/ml with sterile Phosphate Buffered Saline (PBS) and another PBS solution containing of1-ehyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride(EDC) was added into. Then the solution was adjusted to pH7.0and streptavidin with PBS was added to reacte at4℃overnight, streptavidin labeling USPIO-PBCA nanoparticles were made. The molar ratioof USPIO-PBCA: streptavidin:EDC is1:10:25(EDC concentration of0.15g/L, optimaztion).
Using the reaction of biotinylated Fluorescein Isothiocyanate (FITC) and streptavidin-USPIO-PBCA nanoparticles and observed the degree of green fluorescence by fluorescence microscopy to determine and evaluate whether streptavidins were successfully linked to the USPIO-PBCA nanoparticles shell.
The biotinylated FITC was droped into the streptavidin-USPIO-PBCA solution by titration to observe the movement of the fluorescence peak by flow cytometry.
3、The connection of the streptavidin labeled USPIO-PBCA and biotinylated MECA-79monoclonal antibody
5μg corresponding biotinylated MECA-79monoclonal antibody reacted with containing10'streptavidin-labeled USPIO-PBCA suspensions. After incubating for10min, anti-MECA-79-USPIO-PBCA nanoparticles were made.
4、Determination the physical and chemical properties of anti-MECA-79monoclonal antibody labelled USPIO-PBCA particle
The apparent morphological characteristics of the prepared anti-MECA-79-USPIO-PBCA were observed by the transmission electron microscope. The size and distribution of nanoparticles was tested by particle size determination.
II MECA-79monoclonal antibody targeted experiments to fresh lymph nodes in normal rabbit
The selected three normal New Zealand white rabbits were anesthetized to take the popliteal lymph nodes in both sides. These six lymph nodes were rinsed in pre-cooling of saline for3-5times to clean the blood and drifted by absorbent filter paper. Freshly removed lymph nodes were immediately snap-frozen at-7℃and fixed by acetone to perform8μm-cryosection. Unspecific binding sites were inhibited (incubation for one hour at37℃, immediate driftion by PBS overnight at4℃) with5%goat serum albumin (prepared by0. lmol/L PBS).Next, sections were incubated for1hour with biotinylated MECA-79antibody in0.1%goat serum albumin (1:100) at room temperature and dealed with H2O2(prepared by0.1mol/L PBS) to block of endogenous peroxidase. After having been rinsed for three times which lasted for5minutes, these sections were incubated with an extravidin peroxidase conjugate for one hour. The peroxidase-based detection system was employed with3-amino-9-ethylcarbazole (6ml20mg/ml solved in dimethylformamide) as the chromogenic substrate. Specificity of staining was confirmed by means of employing an IgM isotype as a negative control. The experiment was divided into two groups:MECA-79monoclonal antibody for the experimental group, another IgM monoclonal antibody as a control group. Respectively, these two groups were investigated with regard to the specificity of for the antibodies to L-selectin ligand immunohistochemically.
Ⅲ The targeted study Of MECA-79monoclonal antibody labelled USPIO-PBCA nanoparticles to normal rabbits'nodes
1、Experimental groups:18normal rabbits were randomly divided into three groups (n=6).
1) Targeted experimental group:Normal New Zealand white rabbits, injection of15μmol Fe MECA-79monoclonal antibody labeled USPIO-PBCA contrast agent with15μmol Fe through the toe clearance by one side.
2) Control group1:Normal New Zealand white rabbits, injection of USPIO-PBCA contrast agent with15μmol Fe through the toe clearance by one side.
3) Control group2:Normal New Zealand white rabbits, injection of USPIO contrast agent with15μmol Fe through the toe clearance by one side.
2、Experimental procedures:
1) Anesthesia:3%pentobarbital sodium (20mg/Kg) ear vein administration, where appropriate, add a small amount of diazepam to anesthetize animals.
2) Fix:The animals were fixed at a suitable board by supine position, as far as possible symmetry.
3、Scanning equipment and parameters
1) Scanning equipment:GE Signa Excite3.0T Magnetic Resonance Imaging system, with head8-channel quadrature coil.
2) Scan sequences and parameters:coronal T2WI,(TR/TE:5000/85ms echo train length(ETL):16, thickness:2mm, Field of View (FOV):12cm, scan matrix:256×256, number of excitations(NEX):3); axial T2WI2-Dimensional gradient echo (TR/TE:400/24ms, flip of angle:20°, thickness:2mm, FOV:12cm, scan matrix:256×256, NEX:2); axial T1WI spin-echo sequence (TR/TE:400/12ms, thickness:2mm, FOV:12cm, scan matrix:256×256, NEX:2), the above sequences of unenhanced and enhanced scans.
3) Contrast-enhanced scan mode:the contrast agent was injected locally in the toe clearance by one side of the rabbit, after injection, at the time point of the No.1,2,8,12,24,48h, imagingly. When MR scans were completed, the data were transmitted to the post-processing workstation to analyze MRI images.
4、Data processing
Lymph nodes signal intensity and muscles signal intensity was measured by the injected side. The larger region of intrist in the backgroud was selected to measure the standard deviation of the background noise. And signal to noise ratio (SNR) of the lymph nodes were calculation.
5、Statistical analysis SPSS13.0was used as analyzing software.Repetitive measurement ANOVA (statistical method: Bonferroni) analyzed the difference in SNR of the lymph nodes Bonferrom).analyzed the difference in SNR of lymph nodes and museles among different groups and time points.P>0.05was regarded as no statistiea;dofferemee. P<0.05was regarded as statistical significant difference. P<0.05was regarded as statistical significant difference.
6、Pathology examination
After scanning by MR enhancement, all animals were sacrificed to take lymph nodes by injection of the air through the ear vein.10-15%neutral formalin fixed, paraffin-embedded sections (slice thickness1.5μm) were prepared. Two series slices were taken from each specimen to undergo Prussion Blue Staining to observe the lymph nodes'iron content in the experimental group and comparison groups through light microscope, and imaging findings were compared.
Results
I By the determination of Malvern-3000HS laser particle size analyzer, the average particle size of USPIO-PBCA was approximately159nm, the polydispersity index was0.23.
Ⅱ The transmission electron microscope showed that USPIO-PBCA nanoparticles were spherical or spherical-like, with ralertively uniform distribution, smooth surface and no adhesion between the particles. The size of the average particle size was about60-70nm, which suggested that small particle size and high stability of USPIO-PBCA nanoparticles were prepared.
ⅢThe magnetic properties measured by vibrating sample magnetometer showed the prepared USPIO-PBCA nanoparticles were superparamagnetic.
Ⅳ By magnetic resonance T2relaxation time scan, T2values of USPIO-PBCA particles gradually decreased with the increasing of iron concentration, and T2relaxation rate of the samples was about0.156×106mol-1s-1.
Ⅴ The results of Fluorescence microscopy and flow cytometry all suggested that USPIO-PBCA has been successfully linked with streptavidine, with the reactivity of biotinylated monoclonal antibody.
Ⅵ The apparent morphological characteristics of the prepared nanoparticles were observed by scanning electron microscopy:the nanoparticles were round or oval, with even distribution and no adhesion to each other and the particle size of80.0nm. Determination of particle size showed that the distribution of particle size was uniform, with the particle size about of220.3nm and the dispersion coefficient about of0.318.
Ⅶ The results of horseradish peroxidase immunohistochemistry of normal rabbits'fresh lymph nodes in vitro showed that MECA-79monoclonal antibody, had specific targeting to normal lymph nodes in targeted experimental group, ordinary IgM monoclonal antibody had no specific targeting in control group.
Ⅸ MRI scans of normal rabbits'popliteal lymph nodes and the SPSS statistical analysis results showed that:on T2*WI, T2WI sequences, the decreasing degree of the SNR of lymph nodes in the targeted group lower than in the two control groups with statistical difference; In the same group, the SNR values at different time points had statistical difference. The two factors had interaction effect. On T1WI sequences, the SNR values in different groups and different time points all had statistical differences, but the two factors had no interaction effects. The SNR of muscles in different groups and at different time points were no statistical difference.
X The results of pathological examination by Prussian blue staining showed that more blue-stained iron particles existed in the rabbits'lymph nodes in the targeted experimental group, and fewer blue-stained iron particles in the two control groups, especially in the medullas of lymph nodes.
Conclusions
I Under the weak acidic conditions, the targeted and superparamagnetic anti-MECA-79-USPIO-PBCA nanoparticles were successfully prepared. With ralertively uniform size, smooth surface, these particles based the next step animal experiments.
Ⅱ In vitro experiment, MECA-79monoclonal antibodies specifically targeted to the paracortex of lymph nodes in normal rabbits. This suggested that the prepapared anti-MECA-79-USPIO-PBCA nanoparticles could specifically target to rabbits'normal lymph nodes.
Ⅲ The animal experiments in vivo showed that the anti-MECA-79-USPIO-PBCA nanoparticles could specifically target to the normal lymph nodes and the SNR values of lymph nodes decreased on three sequences. More, the results of control experiment showed that the negative enhancement in the targeted group superior to the two groups. The Prussian blue stainning results showed that the more and the broader blue-stainning particles and in the targeted grou. This means that the prepared anti-MECA-79-USPIO-PBCA nanoparticles not only engulfed by Phagocytes in Mononuclear-phagocytic system, but aslo had actively specific targeting to the paracotex of the lymph nodes.
一、研究背景：
淋巴结是哺乳动物重要的免疫器官,淋巴通路是恶性肿瘤转移的重要途径,临床上判断淋巴结是否累及,是肿瘤分期、治疗方案选择、疾病预后判断的重要参考指标。淋巴结活检具有较大价值,但是进行淋巴结深部活检或临床治疗后复查常常比较困难,寻找一种简单、安全、无创的淋巴结检查方法受到广泛的重视。
传统上,判断淋巴结的转移与否常采用测定淋巴结径线的方法,此方法作为常规计算机断层扫描(Computed Tomography, CT)或磁共振成像(Magnetic Resonance Imaging, MRI)对淋巴结的定性诊断价值仍有很大的争议。超声波检查淋巴结血管的变化在鉴别淋巴结的良恶性病变中具有一定的意义,但良恶性淋巴结的征象有较多的重叠,超声的诊断准确率偏低。X线淋巴结造影术方法复杂、费时,有一定的并发症,不能满意显示淋巴结细节等特点。正电子发射计算机体层扫描(Positron Emission Tomography, PET)/CT从分子代谢的角度判断淋巴结的转移情况,但分辨率低,且价格昂贵。
采用对比剂增强的MR淋巴管成像是一种新型无创性的检测方法,目前使用最广泛、较理想的对比剂是超顺磁性氧化铁(Superparamagnetic Iron Oxide, SPIO)类对比剂,这是一种网状内皮系统被动靶向对比剂,能够在T2加权成像(T2-weighted Imaging, T2WT)序列上产生负性增强的作用。直径<30nm的SPIO称为超小超顺磁性氧化铁(Ultrasmall Superparamagnetic Iron Oxide, USPIO),它的MRI增强扫描可以发现位于淋巴结内的局限性转移灶,而与淋巴结是否增大无关。但是USPIO是被淋巴结髓质的吞噬细胞吞噬而显像的,属于被动靶向方式,存在不少问题,可能会有假阴性、假阳性的发生,影响诊断的正确性。
L-选择素(L-selectin)存在于正常的淋巴细胞表面上,其正常功能是作为淋巴细胞到达外周淋巴结归巢的受体。淋巴结的高内皮微静脉(High Endothelial Venules, HEV)内皮细胞上、迷路及髓质中,均有L-选择素配体的高度表达,L-选择素配体的核心抗原是MECA-79,其单抗具有与L-选择素相似的作用,能高度特异性与L-选择素配体高表达区结合,已有文献表明抗MECA-79单抗修饰的超声对比剂可以主动靶向正常淋巴结；聚氰基丙烯酸正丁酯(Polybutylcyanoacrylate, PBCA)以安全无毒、生物相容性好等特点成为目前最受重视的药物载体,那么PBCA经此抗体修饰以后,以它为载体携带超顺磁性物质(比如USPIO)可以制成MR对比剂,这种MR增强扫描应该可以实现主动靶向淋巴结,据我们己知的文献尚未见类似报道。
二、研究目的：
1、在弱酸性的条件下,将USPIO颗粒包被在PBCA纳米球内部,碱性条件下处理之后自动生成羧基,USPIO-PBCA经交联剂与链霉亲和素反应,再与生物素化MECA-79单抗,通过生物素-亲和素系统修饰到USPIO-PBCA纳米微粒外壳上,从而制备L-选择素配体靶向性的USPIO-PBCA纳米微粒；
2、动物体外实验,将生物素化MECA-79单抗在正常兔的新鲜淋巴结上做免疫组化实验检测该抗体的特异性靶向能力；
3、正常新西兰大白兔活体动物实验,将制备出的抗MECA-79-USPIO-PBCA纳米微粒通过足背组织间隙注射的方式做MRI增强扫描,在不同时间点检测对正常兔胭窝淋巴结的定性强化效果,病理检查证实。
方法
一、制备与表征
1、USPIO-PBCA纳米微粒的制备及检测
将氰基丙烯酸正丁酯(Butylcyanoacrylate, BCA)单体用CH2-Cl2稀释(VCH2Cl2:VBCA=3:1),配备酸性条件(pH6.4)的缓冲溶液(KH2PO4-NaOH)50ml,在溶液中融入浓度约为5%非离子表面活性剂Dextran-70,将浓度为11.2mg/ml的USPIO滴加入混合溶液中,搅拌5min,搅拌速率为800rpm。加大搅拌速率至1200rpm,缓慢逐滴滴加1.2%BCA单体入混合液中,持续乳化聚合5h,终止反应后减压抽滤,即得PBCA磁性纳米球稳定混悬液。随后用0.45μm微孔滤膜过滤,将滤液存于4℃冰箱内备用。
Malvern-3000HS激光粒度分析仪测纳米微粒的粒径和分布：取上述USPIO-PBCA混合液透析48h,除去游离的USPIO、Dextran-70,取透析过的USPIO-PBCA溶液用Malvern-3000HS激光粒度分析仪测定粒径及其分布。
透射电镜观察纳米微粒的形态：取少量上述经透析过USPIO-PBCA,在透射电镜下观察USPIO-PBCA大体形态。
振动样品磁强计检测微粒的磁学特性：在室温下,将约0.5mg冻干粉状USPIO-PBCA纳米微粒装入直径1cm的试管,密封后置入振动样品磁强计,逐渐加大磁场强度,并从显示屏观察磁化曲线,直到样品的磁化强度不再增加为止,记录磁化曲线。
磁共振扫描测T2时间及T2弛豫率：取USPIO-PBCA溶液适量,分别加水稀释至铁浓度为0.04、0.06、0.08、0.10、0.12、0.14、0.16mmol/L,在MR机器(3.0T,美国GE公司,Signa EXCIT HD)上扫描,采用8通道头部线圈,层厚3.0mm,间隔0.5mm,扫描序列采用T2map:重复时间(Time of Repetiton,TR)3000ms,回波时间(Time of Echo,TE)20ms、40ms、60ms、80ms,均做冠状位扫描。测定T2值：将原始数据导入ADW4.3工作站,测量USPIO-PBCA的T2弛豫时间值。
计算USPIO-PBCA T2弛豫率：T2弛豫率定义为含铁浓度与1/T2直线方程的斜率。
2、链霉亲和素连接USPIO-PBCA微粒及检测
将获得的初始USPIO-PBCA在碱性条件下(pH=9)作用一个小时,使其表面产生羧基基团；用灭菌磷酸盐缓冲液(Phosphate Buffered Saline, PBS)将USPIO-PBCA稀释至含铁量为0.52μM/ml,,将含1-乙基-3-(3-二甲氨基丙基)-碳二亚胺盐酸盐(1-ehy1-3-(3-dimethyl aminopropyl)carbodiimide hydrochloride, EDC)的PBS溶液加入其中,调整溶液pH至7.0,然后加入链霉亲和素的PBS溶液,4℃反应过夜,得到链霉亲和素标记的USPIO-PBCA纳米微粒。反应摩尔比：USPIO-PBCA:链霉亲和素：EDC为1：10：25(EDC浓度在0.15g/L最佳)。
用生物素化的生物素-异硫氰酸荧光黄(fluorescein isothiocyanate, FITC)与链霉亲和素化的USPIO-PBCA纳米微粒反应,用荧光显微镜观察荧光程度来评价链霉亲和素是否成功接到USPIO-PBCA纳米微粒外壳上
用滴定法将生物素化的FITC逐滴滴加在链霉亲和素化的USPIO-PBCA溶液里,采用流式细胞仪观察荧光峰的移动情况。
3、链霉亲和素标记的USPIO-PBCA与生物素化MECA-79单抗的链接加入5μg的对应生物素化的MECA-79单抗在含有107链霉亲和素标记的USPIO-PBCA的混悬液中,孵育10min,即可得抗MECA-79-USPIO-PBCA纳米微粒。
4、MECA-79单抗标记的USPIO-PBCA微粒理化特性的测定
投射电子显微镜观察已制备纳米微粒的表观形态特征,粒度测定测试MECA-79单抗标记的USPIO-PBCA纳米微粒的分布、粒径。
二、MECA-79单抗在正常兔新鲜淋巴结的靶向性实验
实验分组方法：
1)、实验组：生物素化的MECA-79单抗；
2)、对照组：另外一种生物素化的同源性IgM。
分别作免疫组化,观察抗体的靶向性情况。
选用正常的新西兰大白兔3只,麻醉后每只动物取双侧胭窝淋巴结各1枚,一共6枚,预冷的生理盐水反复漂洗3-5次,漂净血水,吸水滤纸吸干水分,将新鲜的正常淋巴结立刻放入-70℃快速冷冻,丙酮固定,行冰冻切片,片厚8μm,非特异的结合点先用5%(0.1mol/L PBS配制)山羊血清白蛋白进行封闭(37℃,孵育.1小时,立即用PBS漂洗过夜,4℃)；室温下,切片放在含1%生物素化单抗的山羊血清白蛋白(1：100)孵育1小时,再用3%的H2O2(0.1mol/LPBS配制)处理30min,以封闭内源性的辣根过氧化物酶的活性；然后O.1mol/LPBS漂洗3x5min,再滴加一种外源性的亲和素性过氧化物酶孵育1h,显微镜下观察显色情况。
三、MECA-79单抗标记的USPIO-PBCA纳米微粒对正常兔淋巴结的靶向性成像研究
1、实验分组：将18只正常新西兰大白兔随机分为三组,每组6只。
1)靶向组：正常新西兰大白兔,经一侧足趾间隙注射含Fe15μmol的抗MECA-79-USPIO-PBCA对比剂；
2)对照组1：正常新西兰大白兔,经一侧足趾间隙注射含Fe15μmol的USPIO-PBCA对比剂；
3)对照组2：正常新西兰大白兔,经一侧足趾间隙注射含Fe15μmol的USPIO对比剂。
2、实验步骤：
1)麻醉：3%戊巴比妥钠(20mg/Kg)经耳缘静脉给药,酌情添加少量安定,麻醉动物。
2)固定：以仰卧位的方式将动物固定在一合适木板上,左右尽量对称。
3、扫描设备及参数
1)扫描设备：GE Signa Excite3.0T MR磁共振成像系统,采用膝关节线圈。
2)扫描序列及参数：冠状位、矢状位T2WI(TR/TE:5000/85ms,回波链(Echo Train Length, ETL):16,层厚：2mm,视野(Field of View, FOV):12cm,扫描矩阵：256×256,重复次数：3)；冠状位、矢状位T2WI,2D梯度回波T2*加权成像(T2*-Weighted Imaging,T2*WI)(TR/TE:400/24ms,反转角：20-,层厚：2mm, FOV:12cm,扫描矩阵：256×256,脉冲重复激发次数(Number of Excitations, NEX):2);矢状位、横断位T1加权成像(T1-Weighted Imaging,T1WI)自旋回波序列(TR/TE:400/12ms,层厚：2mm, FOV:12cm,扫描矩阵：256×256, NEX：2),以上序列需要平扫及增强扫描。
3)对比剂增强扫描方式：向兔的一侧足趾间隙局部注射对比剂(一侧肢体注射总容量0.2m1),注射后1,2,8,12,24,48h分别成像。MR扫描完成后,将数据传至后处理工作站,进行后续的MRI图像分析。
4、数据处理
分别测定注射对比剂侧的淋巴结信号强度、肌肉组织信号强度,在背景区域选取较大的ROI,测背景噪声的标准差。计算各组淋巴结的信噪比。
5、统计学分析
采用SPSS13.0软件包,采用重复测量的方差分析(统计方法：Bonferroni)对比增强前后各组淋巴结及肌肉组织信噪比(Signal to Noise Ratio, SNR)是否有显著性差异。用单因素方差分析(统计方法：Bonferroni)分析组间不同时间点淋巴结及肌肉组织SNR是否有显著性差异,P<0.05认为有显著性差异。
6、病理学检查
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,取出胭窝淋巴结,然后用10-15%中性福尔马林浸泡、固定,常规石蜡包埋、切片,片厚1.5μm,行普鲁士蓝染色(Prussion Blue Staining),分别观察实验组、对照组1和对照组2淋巴结组织常规光镜下的形态及组织内含Fe的情况,与影像学表现进行对照分析。
结果
一、用Malvern-3000HS激光粒度仪测定的USPIO-PBCA平均粒径约为159nm,多分散系数为0.23。
二、透射电镜下观察USPIO-PBCA纳米微粒呈球形、类球形,分布较均匀,表面光滑、平整,粒子之间无粘连,粒子平均粒径大小约为60-70nm,提示制备出了小粒径、高稳定性的USPIO-PBCA纳米颗粒。
三、振动样品磁强计磁学特性测试结果显示制备出的USPIO-PBCA纳米颗粒具有超顺磁性。
四、经磁共振T2弛豫时间扫描,随着铁浓度的增加,USPIO-PBCA的T2值逐渐下降,所得样品的T2弛豫率0.156x106mol-1.s-1。
五、荧光显微镜和流式细胞仪检测结果,均提示USPIO-PBCA已经成功链霉亲和素化,并且具备与生物素化的单抗反应活性。
六、扫描电子显微镜观察已制备纳米微粒的表观形态特征,显示纳米微粒成球规整,呈圆形或者椭圆形,分布均匀,彼此不粘连,粒径约80nm。粒度测定显示粒度分布均匀,粒径约220.3nm,分散系数为0.318。
七、正常兔新鲜淋巴结辣根过氧化物酶免疫组化结果显示,靶向组MECA-79单抗对正常淋巴结有特异性染色,对照组普通IgM单抗则没有特异性染色。
八、磁共振扫描正常兔胭窝淋巴结和SPSS统计学分析结果显示,在T2*WI、T2WI序列上,靶向实验组的淋巴结SNR降低低于对照组1和对照组2淋巴结,差异有统计学意义,同一组间不同时间点差异也有统计学意义,不同组间和不同时间点有交互效应；在T1WI序列上,三组淋巴结的SNR差异有统计学意义,不同时间点差异也有统计学意义,不同组间和时间点无交互效应；三组肌肉组织SNR在T2*WI、T2WI、T1WI序列上,不同组间及组内不同时间点差异无统计学意义。
九、病理检查普鲁士蓝染色结果显示,靶向组注射侧的兔淋巴结内含有较多蓝染的铁颗粒,分布范围较广,对照组两组内蓝染的铁颗粒相对较少,且主要局限于淋巴结的髓质内。
结论
一、在弱酸性条件下,成功制备出具有超顺磁性的抗MECA-79-USPIO-P BCA靶向性纳米微粒,粒子大小较均匀,表面光滑、平整,为后续动物实验打下基础。
二、MECA-79单抗可以特异性靶向兔的正常淋巴结的副皮质区,可以间接说明制备出的抗MECA-79-USPIO-PBCA纳米微粒可以特异性靶向兔正常淋巴结。
三、动物活体实验表明,抗MECA-79-USPIO-PBCA纳米微粒对正常淋巴结具有特异靶向性,可以使正常淋巴结在T2*WI、T2WI、T1WI上SNR降低；而且对照实验表明靶向组的负性强化作用要明显强于两组对照组,普鲁士蓝染色结果显示靶向组的淋巴结内蓝染的铁颗粒较多,而且范围更广,说明制备出的抗MECA-79-USPIO-PBCA纳米微粒不仅能够被单核-吞噬系统所吞噬,而且能够主动靶向淋巴结的副皮质区。
Background and Objective
Background
Lymph nodes are important immune organs in mammalian and the lymphatic pathway is an important route of tumor metastasis. To judge the lymph nodes involved or not, it is a major index for tumor stage, treatment alternatives, disease prognosis. Although with great values, it has many difficulties in lymph nodes biopsies in deep tissue or review after clinical treatment. Nowadays, to find a simple, safe, non-invasive lymph node examination method has been received wide attentions.
Traditionally, to judge lymph node metastasis or not, it was often to use the method of determination of lymph node size. But, in the qualitative diagnostic value of lymph node, this method still has considerable controversy in routine Computed Tomography (CT) or Magnetic Resonance Imaging (MRI) scan. Ultrasound examing the vascular change of lymph node has some significance in the identification of the benign or malignant lesions in lymph nodes. Because of the overlap signs between in the benign and the malignant lymph nodes, the diagnostic accuracy of ultrasound is ralertively low. X-ray lymphadenography are complex, time-consuming, with some complications and can not satisfactorily display the details of lymph nodes. PET (Positron Emission Tomography)/CT could determine the lymph node metastasis from the perspective of molecular metabolism, but the resolution is low.
Contrast-enhanced MR lymphatic imaging is a new noninvasive detection method. Currently the Superparamagnetic Iron Oxides (SPIO)s contrast agents are the most widely used and the preferred ones which are a kind of passively targeting to reticuloendothelial system contrast agents with negative enhancement effect on the T2WI sequences. Ultrasmall Superparamagnetic Iron Oxides (USPIO)(the diameter less than30nm in SPIO) enhancement MRI scans can find localized metastases in the lymph nodes independent of the sizes of lymph nodes. But USPIO particles were engalfed by the phagocytic cells in the lymph node medulla to image. This enhancement MRI is a passive targeting method with many problems, which may cause false positives and false negtives and affect the accuracy of diagnosis.
L-selectins exit in the surface of normal lymphocytes, as the homing receptors which lead peripheral lymphocytes to reach lymph nodes. L-selectin ligands highly express in the endothelial cells of high endothelial veins, labyrinths and medullas of lymph nodes and its core antigens are MECA-79monoclonal antibodies, which have a similar role with L-selectin and highly specific to combine with the areas where L-selectin ligand highly expressed in. Literature suggested that ultrasound contrast agent modified by MECA-79monoclonal antibody could target initiatively to normal lymph nodes. Therefore, superparamagnetic substances (for example USPIO) carried by the antibody may target initiatively to lymph nodes in MR enhancement scan. Being of safe and good biocompatibility characteristics, Polybutylcyanoacrylate (PBCA) was the most important drug carrier. So, after PBCA were modified by this antibody, these nanoparticles which carryed superparamagneticsubstances (such as USPIO) could be prepared as MR contrast agent, this enhanced MR scan should be able to target to the lymph nodes actively. As we known that no similar literatures have reported.
Objectives of Study
1、In the weakly acidic conditions, the USPIO particles were coated in the PBCA nanospheres. After processing under alkaline conditions to generate carboxyls automatically, USPIO-PBCA particles reacted with streptavidins by crosslinking agent. Then, biotinylated MECA-79monoclonal antibodies were modified to USPIO-PBCA nanoparticles shells by biotin-avidin system, which made nanoparticles initiativly targeting to L-selectin ligands of lymph nodes.
2、The animal in vitro experiments:the specific targeting abilities of the biotinylated MECA-79monoclonal antibodies were detected by immunohistochemistry in fresh lymph nodes of normal rabbits.
3、Normal live New Zealand white rabbits experiments:by injection of anti-MECA-79-of USPIO-PBCA nanoparticles through the dorsal gap injection, the qualitative enhancement on normal rabbit popliteal lymph nodes at different time points by MRI scan, then by confirmed in pathology.
Methods
I Preparation and characterization
1、Synthesis and detection of USPIO-PBCA nanoparticles
BCA monomer was diluted by CH2-Cl2(VCH2ci2:VBCA=3:1).The non-ionic surfactant of5%Dextran-70was dissolved into the acidic (pH6.4) buffer solution (KH2PO4-NaOH) solution and the concentration of11.2mg/ml USPIO was dropped to the mixed solution of50ml. Afer stirring5minutes with the stirring speed of800rpm to1200rpm incresingly,1.2%concentration of the butylcyanoacrylate (BCA) monomer was slowly added into the mixture solution drop by drop. After emulsion polymerization continuously for5hours, the reaction was terminated. After this solution was filtered by vacuum pumb, the stable suspension of PBCA magnetic nanoparticles was prepared. Following filtred by0.45μm microporous filtration membrane, the filtrate was kept in the refrigerator of4℃for use.
Size and distribution of particle nanoparticles:Malvern-3000HS laser particle size analyzer: Take the above of USPIO-PBCA mixture to dialysis for48hours to remove of free USPIO and Dextran-70. The dialysised USPIO-PBCA solution was detected about the size and distribution by Malvern-3000HS laser particle size analyzer.
The morphology of nanoparticles by transmission electron microscopy:Take a small amount of the above of dialysised USPIO-PBCA, then the gross shape of it was observed by transmission electron microscopy.
The measured T2relaxation time and the calculated T2relaxation rate by Magnetic resonance scan:appropriate amount of USPIO-PBCA solution was diluted by water to the iron concentration of0.04,0.06,0.08,0.10,0.12,0.14,0.16mmol/L respectively, then scanned in the MR machine (3.0T U.S. company GE, Sigma EXCIT HD) with an8-channel head coil, thickness of3.0mm, interval of0.5mm, the scanning sequence T2map:time of repetion (TR) of3000ms, time of echo(TE) of20ms,40ms,60ms,80ms, all with a coronal scan. Determination of T? values: raw data were imported to ADW4.3workstation and USPIO-PBCA T2relaxation time values were measured.
Calculate the T2relaxation rate of USPIO-PBCA:T2relaxation rate is defined as the slope of the linear equation about the iron concentration and1/T2.
2、Preparation and detection Streptavidin-linked USPIO-PBCA particles
The prepared USPIO-PBCA particles were dealed with under alkaline conditions (pH=9) for an hour, so carboxyl groups generated on the surfaces of particles. Thses USPIO-PBCA particles were diluted to the iron concentration of USPIO-PBCA0.52μM/ml with sterile Phosphate Buffered Saline (PBS) and another PBS solution containing of1-ehyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride(EDC) was added into. Then the solution was adjusted to pH7.0and streptavidin with PBS was added to reacte at4℃overnight, streptavidin labeling USPIO-PBCA nanoparticles were made. The molar ratioof USPIO-PBCA: streptavidin:EDC is1:10:25(EDC concentration of0.15g/L, optimaztion).
Using the reaction of biotinylated Fluorescein Isothiocyanate (FITC) and streptavidin-USPIO-PBCA nanoparticles and observed the degree of green fluorescence by fluorescence microscopy to determine and evaluate whether streptavidins were successfully linked to the USPIO-PBCA nanoparticles shell.
The biotinylated FITC was droped into the streptavidin-USPIO-PBCA solution by titration to observe the movement of the fluorescence peak by flow cytometry.
3、The connection of the streptavidin labeled USPIO-PBCA and biotinylated MECA-79monoclonal antibody
5μg corresponding biotinylated MECA-79monoclonal antibody reacted with containing10'streptavidin-labeled USPIO-PBCA suspensions. After incubating for10min, anti-MECA-79-USPIO-PBCA nanoparticles were made.
4、Determination the physical and chemical properties of anti-MECA-79monoclonal antibody labelled USPIO-PBCA particle
The apparent morphological characteristics of the prepared anti-MECA-79-USPIO-PBCA were observed by the transmission electron microscope. The size and distribution of nanoparticles was tested by particle size determination.
II MECA-79monoclonal antibody targeted experiments to fresh lymph nodes in normal rabbit
The selected three normal New Zealand white rabbits were anesthetized to take the popliteal lymph nodes in both sides. These six lymph nodes were rinsed in pre-cooling of saline for3-5times to clean the blood and drifted by absorbent filter paper. Freshly removed lymph nodes were immediately snap-frozen at-7℃and fixed by acetone to perform8μm-cryosection. Unspecific binding sites were inhibited (incubation for one hour at37℃, immediate driftion by PBS overnight at4℃) with5%goat serum albumin (prepared by0. lmol/L PBS).Next, sections were incubated for1hour with biotinylated MECA-79antibody in0.1%goat serum albumin (1:100) at room temperature and dealed with H2O2(prepared by0.1mol/L PBS) to block of endogenous peroxidase. After having been rinsed for three times which lasted for5minutes, these sections were incubated with an extravidin peroxidase conjugate for one hour. The peroxidase-based detection system was employed with3-amino-9-ethylcarbazole (6ml20mg/ml solved in dimethylformamide) as the chromogenic substrate. Specificity of staining was confirmed by means of employing an IgM isotype as a negative control. The experiment was divided into two groups:MECA-79monoclonal antibody for the experimental group, another IgM monoclonal antibody as a control group. Respectively, these two groups were investigated with regard to the specificity of for the antibodies to L-selectin ligand immunohistochemically.
Ⅲ The targeted study Of MECA-79monoclonal antibody labelled USPIO-PBCA nanoparticles to normal rabbits'nodes
1、Experimental groups:18normal rabbits were randomly divided into three groups (n=6).
1) Targeted experimental group:Normal New Zealand white rabbits, injection of15μmol Fe MECA-79monoclonal antibody labeled USPIO-PBCA contrast agent with15μmol Fe through the toe clearance by one side.
2) Control group1:Normal New Zealand white rabbits, injection of USPIO-PBCA contrast agent with15μmol Fe through the toe clearance by one side.
3) Control group2:Normal New Zealand white rabbits, injection of USPIO contrast agent with15μmol Fe through the toe clearance by one side.
2、Experimental procedures:
1) Anesthesia:3%pentobarbital sodium (20mg/Kg) ear vein administration, where appropriate, add a small amount of diazepam to anesthetize animals.
2) Fix:The animals were fixed at a suitable board by supine position, as far as possible symmetry.
3、Scanning equipment and parameters
1) Scanning equipment:GE Signa Excite3.0T Magnetic Resonance Imaging system, with head8-channel quadrature coil.
2) Scan sequences and parameters:coronal T2WI,(TR/TE:5000/85ms echo train length(ETL):16, thickness:2mm, Field of View (FOV):12cm, scan matrix:256×256, number of excitations(NEX):3); axial T2WI2-Dimensional gradient echo (TR/TE:400/24ms, flip of angle:20°, thickness:2mm, FOV:12cm, scan matrix:256×256, NEX:2); axial T1WI spin-echo sequence (TR/TE:400/12ms, thickness:2mm, FOV:12cm, scan matrix:256×256, NEX:2), the above sequences of unenhanced and enhanced scans.
3) Contrast-enhanced scan mode:the contrast agent was injected locally in the toe clearance by one side of the rabbit, after injection, at the time point of the No.1,2,8,12,24,48h, imagingly. When MR scans were completed, the data were transmitted to the post-processing workstation to analyze MRI images.
4、Data processing
Lymph nodes signal intensity and muscles signal intensity was measured by the injected side. The larger region of intrist in the backgroud was selected to measure the standard deviation of the background noise. And signal to noise ratio (SNR) of the lymph nodes were calculation.
5、Statistical analysis SPSS13.0was used as analyzing software.Repetitive measurement ANOVA (statistical method: Bonferroni) analyzed the difference in SNR of the lymph nodes Bonferrom).analyzed the difference in SNR of lymph nodes and museles among different groups and time points.P>0.05was regarded as no statistiea;dofferemee. P<0.05was regarded as statistical significant difference. P<0.05was regarded as statistical significant difference.
6、Pathology examination
After scanning by MR enhancement, all animals were sacrificed to take lymph nodes by injection of the air through the ear vein.10-15%neutral formalin fixed, paraffin-embedded sections (slice thickness1.5μm) were prepared. Two series slices were taken from each specimen to undergo Prussion Blue Staining to observe the lymph nodes'iron content in the experimental group and comparison groups through light microscope, and imaging findings were compared.
Results
I By the determination of Malvern-3000HS laser particle size analyzer, the average particle size of USPIO-PBCA was approximately159nm, the polydispersity index was0.23.
Ⅱ The transmission electron microscope showed that USPIO-PBCA nanoparticles were spherical or spherical-like, with ralertively uniform distribution, smooth surface and no adhesion between the particles. The size of the average particle size was about60-70nm, which suggested that small particle size and high stability of USPIO-PBCA nanoparticles were prepared.
ⅢThe magnetic properties measured by vibrating sample magnetometer showed the prepared USPIO-PBCA nanoparticles were superparamagnetic.
Ⅳ By magnetic resonance T2relaxation time scan, T2values of USPIO-PBCA particles gradually decreased with the increasing of iron concentration, and T2relaxation rate of the samples was about0.156×106mol-1s-1.
Ⅴ The results of Fluorescence microscopy and flow cytometry all suggested that USPIO-PBCA has been successfully linked with streptavidine, with the reactivity of biotinylated monoclonal antibody.
Ⅵ The apparent morphological characteristics of the prepared nanoparticles were observed by scanning electron microscopy:the nanoparticles were round or oval, with even distribution and no adhesion to each other and the particle size of80.0nm. Determination of particle size showed that the distribution of particle size was uniform, with the particle size about of220.3nm and the dispersion coefficient about of0.318.
Ⅶ The results of horseradish peroxidase immunohistochemistry of normal rabbits'fresh lymph nodes in vitro showed that MECA-79monoclonal antibody, had specific targeting to normal lymph nodes in targeted experimental group, ordinary IgM monoclonal antibody had no specific targeting in control group.
Ⅸ MRI scans of normal rabbits'popliteal lymph nodes and the SPSS statistical analysis results showed that:on T2*WI, T2WI sequences, the decreasing degree of the SNR of lymph nodes in the targeted group lower than in the two control groups with statistical difference; In the same group, the SNR values at different time points had statistical difference. The two factors had interaction effect. On T1WI sequences, the SNR values in different groups and different time points all had statistical differences, but the two factors had no interaction effects. The SNR of muscles in different groups and at different time points were no statistical difference.
X The results of pathological examination by Prussian blue staining showed that more blue-stained iron particles existed in the rabbits'lymph nodes in the targeted experimental group, and fewer blue-stained iron particles in the two control groups, especially in the medullas of lymph nodes.
Conclusions
I Under the weak acidic conditions, the targeted and superparamagnetic anti-MECA-79-USPIO-PBCA nanoparticles were successfully prepared. With ralertively uniform size, smooth surface, these particles based the next step animal experiments.
Ⅱ In vitro experiment, MECA-79monoclonal antibodies specifically targeted to the paracortex of lymph nodes in normal rabbits. This suggested that the prepapared anti-MECA-79-USPIO-PBCA nanoparticles could specifically target to rabbits'normal lymph nodes.
Ⅲ The animal experiments in vivo showed that the anti-MECA-79-USPIO-PBCA nanoparticles could specifically target to the normal lymph nodes and the SNR values of lymph nodes decreased on three sequences. More, the results of control experiment showed that the negative enhancement in the targeted group superior to the two groups. The Prussian blue stainning results showed that the more and the broader blue-stainning particles and in the targeted grou. This means that the prepared anti-MECA-79-USPIO-PBCA nanoparticles not only engulfed by Phagocytes in Mononuclear-phagocytic system, but aslo had actively specific targeting to the paracotex of the lymph nodes.
引文
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