摘要
恶性肿瘤已经成为威胁人类健康的最重要疾病之一,而对肿瘤诊断的分期对临床治疗方案的制定及病人预后具有重大的意义,淋巴结的局部和远处转移是恶性肿瘤特别是上皮类恶性肿瘤的重要转移途径,如何正确判断恶性肿瘤病人体内淋巴结是否转移是当前研究的重点和要点。现在临床上常规使用的超声、CT、MR及PEC/CT对于淋巴结的判断具有一定的局限,容易造成误诊或漏诊已转移的淋巴结,因此,寻找一种新的方法当务之急,MR虽具有较高的软组织分辨率,可很好的检测出淋巴结,但是由于临床常规Gd-DTPA造影成像与CT增强相似,其对淋巴结内微小转移灶特异性和敏感性较低,超顺磁性氧化铁负性对比剂是目前研究的重点,目前已经在国外运用于临床的USPIO纳米粒,明显提高了对转移性淋巴结的诊断率,其是一种在体内被内皮网状细胞被动摄取的对比剂,注射入体内后,循环到淋巴结时被其髓质内的巨噬细胞吞噬,从而导致负性增强效果。转移性淋巴结由于被增生的肿瘤占据,不具备巨噬细胞,因此注射USPIO造影剂后信号不发生改变或仅局部信号下降,可提示其为转移性肿瘤。肿瘤细胞进入淋巴结首先通过淋巴结的被膜下窦,部分沉积于此并增生,在此生长并形成转移灶,而反应增生性淋巴结常常有皮质及皮质旁增生导致淋巴结皮质的增厚,巨噬细胞主要集中在淋巴结的髓质内,被吞噬的USPIO产出的磁化率效应,表现为淋巴结中心的低信号,而转移性一般表现为偏心性的高信号。L-选择素(核心抗原结构式MECA-79或称为外周淋巴结血管定居素,PNAd)配体,该结构表达于淋巴结高血管内皮、淋巴结迷路中,MECA-79单抗具有与L-选择素相似的作用,能特异性与高内皮微静脉、淋巴迷路等L-选择素配体表达区结合,可作为携带超顺磁性物质高度靶向淋巴结正常结构的探针,在前期工作中,已经合成了抗MECA-79-PBCA-USPIO纳米颗粒并成功靶向淋巴结,本研究的主要目的是优化制备具有特异性靶向淋巴结的抗MECA-79-PBCA-USPIO纳米颗粒的制备工艺、对其表征并在动物模型上考察其对良恶性淋巴结的诊断效果。
目的
1、优化抗MECA-79-PBCA-USPIO纳米微粒制备工艺,并对其终产物进行表征,在体外评价其对细胞的毒性作用；
2、在动物模型上初步评价抗MECA-79-PBCA-USPIO对良恶性淋巴结的鉴别诊断价值。
方法
一、抗MECA-79-PBCA-USPIO纳米微粒的制备优化、表征及体外细胞毒性检测
1三步法合成抗MECA-79-PBCA-USPIO
共沉淀法合成超小超顺磁性氧化铁纳米颗粒(USPIO),用BCA单体包裹USPIO形成PBCA-USPIO纳米颗粒并其在表面修饰制备出抗MECA-79-PBCA-USPIO。
1.1采用共沉淀法,在碱性条件下与月桂酸反应合成月桂酸稳定的USPIO纳米微粒。
1.2对所合成工艺采用正交析因设计进行处理优化其合成主体PBCA-USPIO的影响因素dextran-70浓度、BCA浓度、PH值等对合成PBCA的影响,选择最佳的合成条件。
1.3将优化条件下获得的PBCA-USPIO碱化1h使纳米粒表面产生羧基,然后通过EDC作用与链霉亲和素上氨基形成稳固的酰胺键,链霉亲和素化的PBCA-USPIO纳米溶液中加入生物素化的抗MECA-79单抗,通过生物素-亲和素系统使抗MECA-79单抗结合到PBCA-USPIO上形成我们需要的抗MECA-79-PBCA-USPIO纳米颗粒。
2PBCA-USPIO、抗MECA-79-PBCA-USPIO纳米微粒的表征
2.1PBCA-USPIO、抗MECA-79-PBCA-USPIO纳米微粒在Malvern-3000HS激光粒度分析仪检测水合粒径及分散度取上述两种纳米微粒悬浊液1ml,稀释后置于激光散射粒度分析仪中测试,所选激光光源波长为633.0nm,测试温度为25.00±0.05℃,经样品散射后的光是在90°范围内变化不同的角度来测定的。光信号用256通道、高速数字相关器进行处理。
2.2PBCA-USPIO、抗MECA-79-PBCA-USPIO在透射电镜下观察形态。取少量的PBCA-USPIO及抗MECA-79-PBCA-USPIO纳米粒悬浊液稀释后滴少许于铜网上,待干燥后用滴加磷钨酸负染,静置充分干燥,置于透射显微镜下观察。
2.3PBCA-USPIO、抗MECA-79-PBCA-USPIO纳米微粒的X-ray粉末衍射分析将冷冻干燥后两种纳米粒取少量置于研磨皿中研磨呈粉末状,置于X-ray衍射仪中检测,将所得X-ray图与Fe3O4标准图相比较,确定其具备的晶体结构。
2.4抗MECA-79-PBCA-USPIO在HH-15振动样品磁强计测定其磁学特征精密称取一定量经真空干燥后的抗MECA-79-PBCA-USPIO纳米微粒,置于HH-15振动样品磁强计中,绘制其磁滞曲线并测定该纳米粒的磁饱和强度。
2.5抗MECA-79-PBCA-USPIO的T2磁豫率测定称取一定量的抗MECA-79-PBCA-SPIO纳米粒悬浊液适量,加水稀释成铁浓度为0.04、0.06、0.08、0.10、0.12、0.14和0.16mmol/L的溶液,分装于7支试管中,进行T2map序列扫描,后处理得出的T2弛豫时间后可计算出所得纳米悬浊液的T2磁豫率。
3.体外评价抗MECA-79-PBCA-USPIO的细胞毒性先将正常肝细胞(LO2)复苏培养,LO2细胞复苏后常规传代培养后,以5×103个细胞/孔接种于96孔板上,培养24h后,使用不同浓度的裸SPIO及抗MECA-79-PBCA-USPIO与正常肝细胞孵化24h,MTT法检测其细胞生存率。
二、抗MECA-79-PBCA-USPIO鉴别良恶性淋巴结的MRI研究
(一)实验对象
实验动物：健康成年新西兰白兔24只,体重2.0-2.5kg,雌雄不限,采用随机法分为2组建立模型：淋巴结炎性反应增生组(n=12)：用于建立蛋黄乳胶刺激后淋巴结反应性增生模型；肿瘤转移性淋巴结组(n=12)：用于VX2肿瘤建立淋巴结转移瘤模型。
(二)动物模型建立
1、淋巴结反应性增生组动物模型的建立
新西兰大白兔(12只)麻醉完毕后,在其一侧后肢股外直肌区,注射约0.5ml的蛋黄溶液(生理盐水：蛋黄=1：1),3-4天后重上述操作一次,注射后3-4天即可在胭窝处触及1cm左右的肿大淋巴结,淋巴结反应性增生模型建成。
2、淋巴结VX2肿瘤转移组模型的建立：
新西兰大白兔(12只)进行麻醉后,用注射器吸取约0.5ml所制备得VX2肉瘤组织悬液肿瘤注射接种至一侧后肢外直肌区,2-3周后接种后腿肿块形成,可触及胭窝淋巴结,VX2肉瘤淋巴结转移模型造模建成。
(三)、MRI扫描
1、平扫：将模型动物麻醉后,选用专用小动物兔线圈,扫描序列及参数为：T1WI(TR/TE:633/20ms,NEX:2),脂肪抑制T2WI(TR/TE=5000/65ms, NEX:2),T2WI(TR/TE=6860/62ms,NEX:2).T2*WI(TR/TE=450/12ms,翻转角=20°,NEX:2).以冠状位和矢状位扫描为主,FOV=160mm,层厚=1.5mm,层间距=0mm。
2、增强扫描
(1)反应性增生淋巴结组：采用随机法将反应性增生淋巴结组的新西兰白兔分成SPIO组增强Ⅰ组(Ⅱ=6)和抗MECA-79-PBCA-USPIO组增强Ⅱ组(n=6)。在平扫完毕后,在增强Ⅰ组新西兰白兔淋巴结增大侧后脚掌注射20umol Fe SPIO,在增强Ⅱ组注射20umol Fe抗MECA-79-PBCA-USPIO,扫描时间点为给药后第12小时,扫描参数同平扫；
肿瘤淋巴结转移增生组：采用随机法将淋巴结转移组新西兰白兔分成SPIO组增强Ⅰ组(n=6)和抗MECA-79-PBCA-USPIO组增强Ⅱ组(n=6)。在平扫完毕后,在增强Ⅰ组新西兰白兔淋巴结增大侧后脚掌注射20umol Fe SPIO,在增强Ⅱ组注射20umol Fe抗MECA-79-PBCA-USPIO,扫描时间点为给药后第12小时,扫描参数同平扫。
3、图像评估
1、测量淋巴结大小
在MRI后处理工作站中测量淋巴结大小,T1WI像上,测量各组胭窝淋巴结的长径和短径,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。
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胭窝淋巴结的信号强度,计算各组淋巴结的信噪比(Signal to noise ratio SNR),评价增强扫描后SPIO、抗MECA-79-PBCA-USPIO对鉴别良恶性淋巴结的准确性。
3、病理组织学检查
所有实验动物在完成MRI增强扫描后,在麻醉情况下,用空气注入法处死动物,仔细暴露胭窝淋巴结,摘除并测量淋巴结的长径和短径,计算淋巴结数目。然后用10%中性福尔浸泡、固定,石蜡包埋,行H-E染色及普鲁士蓝染色,以观察胭窝淋巴结炎性反应增生及肿瘤浸润情况,病理组织学与MRI图像相对照。
结果
1、正交实验析因分析及极差分析结果显示(F=1595.667,P=0.001),选择制备抗MECA-79-PBCA-USPIO纳米粒的最佳合成工艺是dextran-70浓度为15%、BCA浓度为3%、PH值为6.0。
2、Malvern激光粒度分析仪测定PBCA-USPIO、抗MECA-79-PBCA-USPIO的强均粒径和多分散系数分别为90nm和0.20、96nm和0.34nm,证明制备纳米粒径<100nm,分散性好。
3、透射电镜下PBCA-USPIO、抗MECA-79-PBCA-USPIO纳米微粒的形态大小均匀,呈球形,包壳完整,可见壳-核结构,部分纳米粒聚集,测量得出PBCA-USPIO、抗MECA-79-PBCA-USPIO粒径约为25±5nm,30±5nm。
4、PBCA-USPIO、抗MECA-79-PBCA-USPIO纳米微粒样本的X-ray衍射各特征峰位置与Fe304晶体标准X-ray图谱中的位置完全一致,证明了我们最终合成的PBCA-USPIO、抗MECA-79-PBCA-USPIO纳米微粒经各种反应过程对其中的Fe304晶型没有影响,符合超顺磁性氧化铁的要求。
5、抗MECA-79-PBCA-USPIO具有超顺磁性,抗MECA-79-PBCA-USPIO质量饱和磁化强度分别为22emu/gFe。
6、抗MECA-79-PBCA-USPIO纳米微粒的弛豫率结果显示抗MECA-79-PBCA-USPIO的T2弛豫率为0.159×106mol-1s-1。
7、体外细胞毒性实验证明了所制备的抗MECA-79-PBCA-USPIO对细胞毒性较低,当其达到1000ug/ml时,细胞生存率约为70%,证明其具有良好的生物相容性及安全性。
8、MRI平扫各不同序列难以准确鉴别炎性增生淋巴和恶性肿瘤转移性淋巴结。
9、与SPIO相似,抗MECA-79-PBCA-USPIO能通过组织间隙注射来进行MRI淋巴结成像。
10、抗MECA-79-PBCA-USPIO纳米微粒能较准确的诊断炎性淋巴结和转移性淋巴结,正确诊断率约为91.7%,高于SPIO组,其诊断正确率为83.3%。
结论
1、通过正交析因分析,确定了制备超顺磁性USPIO-PBCA纳米微粒的最佳条件,保证了PBCA-USPIO合成的纳米粒径,合成的PBCA-USPIO颗粒大小尚均匀,呈圆形或类圆形,表面光整,液态分散性好,电镜下粒径约为25-30nm,流水粒径约90nm, X-Ray衍射显示其具备Fe304晶体结构。
2、通过链霉亲和素-生物素系统,成功制备出抗MECA-79-USPIO-PBCA靶向性对比剂,且具有超顺磁性,质量饱和磁化强度约为22emu/g,经过X-Ray衍射得出其Fe304晶体良好,最终所得抗MECA-79-USPIO-PBCA纳米流体粒径约为96nm,其弛豫率为0.159×106mol-1s-1。
3、抗MECA-79-PBCA-USPIO纳米微粒对细胞毒性小,生物安全性高,生物相容性好,为临床使用提供了安全保障。
4、MRI平扫各序列难以准确鉴别炎性增生淋巴和恶性肿瘤转移性淋巴结；
5、抗MECA-79-PBCA-USPIO能较准确的诊断炎性淋巴结和转移性淋巴结,正确诊断率约为91.7%。 SPIO组诊断正确率为83.3%。
Malignant tumor is one of the most common diseases that threaten human health, the stage of the tumor diagnosis has implications for clinical program development and patient prognosis, local and distant metastasis of lymph nodes is the main pathway of epithelial malignant route of metastasis, how to properly judge the malignancy patient's lymph node metastasis is the current focus of the study.Routine clinical use as ultrasound, CT, MR, and PET/CT for lymph nodes judgment has some limitations, which may cause misdiagnosed lymph node metastasis, therefore, to find a new method urgent.MR has a higher soft tissue resolution that can detected lymph nodes well, but Gd-DTPA contrast enhanced imaging similar to CT enhanced in clinical routine, its specificity and sensitivity still poor, ultrasmall superparamagnetic iron oxide contrast agent has been studied now.Currently the USPIO nanoparticles has applied to the clinical abroad, obviously improve the diagnosis of metastatic lymph nodes.The passive uptake of the contrast agent in the body when injected into the body, it could circulate to the lymph nodes's medullary sinus and phagocytosis by macrophage, resulting in a negative enhancing. Due to metastatic lymph nodes does not have a macrophage, therefore when inject USPIO contrast agent to patient the signal of the lymph nodes does not change or only local signal falls down may be prompted for metastatic lymph nodes. The cells of tumor transfer into the lymph nodes firstly in lymph nodes subcapsular sinus then may be deposited there and hyperplasia and then substitute normal lymph node. while the cortex and cortical hyperplasia in reactive hyperplastic lymph nodes often lead to thickening of the lymph node cortex, USPIO swallow by macrophage that lymph nodes display low signal in lymph node center. Metastatic lymph nodes general performance the localization high signal. L-selectin Which core antigen is MECA-79or called peripheral lymph node vascular addressin, has express in high vascular endothelium, labyrinth in the lymph nodes. MECA-79monoclonal antibody similar to L-selectin ligand that specifically to high endothelial venules, labyrinth in lymph nodes. L-selectin can be used as carrier for superparamagnetic nanopartilces to targeted lymph node. Anti-MECA-79-PBCA-USPIO have been synthesized and successfully targeted lymph nodes nanoparticles in previous work, the purpose of this study is to optimize prescription process and characterize anti-MECA-79-PBCA-USPIO nanoparticles and to differentiate benign and malignant lymph nodes in lymph node model.
Purpose
1、To synthesize anti-MECA-79-PBCA-USPIO nanoparticles and optimize its prescription process, characterize the property of the nanoparticles and evaluate its toxic effects on the cells in vitro;
2、preliminary evaluation of anti-MECA-79-PBCA-USPIO differentiate benign and malignant lymph node in animal models.
Method
1、Optimize prescription process, characterize of Anti-MECA-79-PBCA-USPIO nanoparticles and evaluate cytotoxicity in vitro
1、A three-step synthesis of anti-MECA-79-PBCA-USPIO
At first, ultrasmall superparamagnetic iron oxide nanoparticles be synthesized by coprecipitation, then use the BCA monomer wrap USPIO formate the PBCA-USPIO nanoparticles and prepared anti-MECA-79-PBCA-USPIO by surface modification.
1.1Use coprecipitation reaction under alkaline conditions with lauric Synthesis lauric stable USPIO nanoparticles.
1.2The synthesis process using orthogonal analysis to the optimize process of the main influence factors of PBCA-USPIO, dextran-70concentration, the BCA concentration, pH value on the synthesis of PBCA-USPIO。
1.3It will be obtained under optimized conditions the PBCA-USPIO alkalizate1h Then nanoparticles surface generate carboxyl amino form stable amide bond, and through the EDC role with streptavidin-biotin, the streptavidin and biotinylated the PBCA-USPIO solution adding biotinylated anti-MECA-79monoclonal antibody, anti-MECA-79monoclonal antibody binds by biotin-avidin system so we obtain anti-MECA-79-PBCA-USPIO nanoparticles.
2、Characterizate of PBCA-USPIO, anti-MECA-79-PBCA-USPIO nanoparticle
2.1The size and dispersity of PBCA-USPIO, anti-MECA-79-PBCA-USPIO nanoparticles be test under Malvern-3000HS laser particle size analyzer Take the two nanoparticle suspension1ml dilution then placed in the test on laser light scattering particle size analyzer, the selected wavelength of the laser light source is633.0nm, the test temperature is25.00+0.05℃, the sample scattered light at90°range varied within different angles. The optical signal with256-channel, high-speed digital correlator for processing.
2.2The morphology of PBCA-USPIO and anti-MECA-79-PBCA-USPIO was observed under the transmission electron microscope. Few drops PBCA-USPIO,anti-MECA-79-PBCA-USPIO nanoparticles suspension diluted into copper grids, after drying with phosphotungstic acid negative staining, standing fully dried, observed under the transmission electron microscope.
2.3X-ray diffraction analysis the PBCA-USPIO and anti MECA-79-PBCA-USPIO nanoparticles
Two kinds of nanoparticles after freeze-drying, taking a small amount placed in a grind to powder, placed D/Max-ⅢAX-ray powder diffractometer (polycrystalline) detection of the resulting X-ray diagram with Fe3O4standard chart compared to determine the crystal structure comprising.
2.4HH-15vibrating magnetometer be uesed to measure the characteristics of the anti-MECA-79-PBCA-USPIO
A small quantity of anti-MECA-79-PBCA-USPIO nanoparticles be vacuum drying, then placed in the HH-15vibrating sample magnetometer (VSM) to determinate the change in intensity of the magnetic saturation of nanoparticles, and draw the hysteresis curve.
2.5T2relaxvity of anti MECA-79-PBCA-USPIO nanoparticles
Take a small quantity of anti-MECA-79-PBCA-SPIO nanoparticles suspension be diluted with water in the iron concentration of0.04,0.06,0.08,0.10,0.12,0.14and0.16mmol/L, then put in to seven test tubes and scanned with MR using T2map sequence.Then average T2values of each samples were measured on post-processed,T2relaxvity was calculated
3、In vitro evaluate of the anti-MECA-79-PBCA-USPIO cytotoxicity
After the liver cells (LO2) recovery in culture, Then the cell were seeded in96-well and continued incubating with full media containing uncoated SPIO and anti-MECA-79-PBCA-USPIO with different concentrations respectively. After24h incubation,the viability of the LO2cells was determined by MTT assay
2、Differentiate benign and malignant lymph node by anti-MECA-79-PBCA-USPIO in animal models preliminary
1、Animal
(1) Experimental animals:24healthy adult New Zealand White Rabbits weighted at2.0-2.5kg were used, male or female, were divided into2groups randomly:The reactive hyperplasia group (n=12):the yolk emulsion was used to establish lymph node reactive hyperplasia model; The metastatic lymph node group (n=12):VX2tumor was used to establish lymph node metastases model
(2) The development of model.
1, The reactive hyperplasia lymph nodes group:
New Zealand rabbits anesthesia firstly, then inject0.5ml yolk solution (saline: yolk=1:1) at its side hind limb rectus femoris area, after a repeated injection3days later, the model was successfully created after3days. the swollen lymph node in the popliteal fossa can touch.then reactive hyperplasia lymph node model be builded.
2, The metastasis lymph node group model:
New Zealand white rabbits were anesthetized then inject approximately0.5ml prepared VX2sarcoma tissue suspension tumors at its side hind limb rectus femoris area,2-3weeks later that can touched popliteal lymph node, the metastasis lymph node model be builded.
2、MR imaging
1. Pre-scan The model animals were anesthetized, choose the small animal rabbit coil. All the MR imaging was performed with sequences and parameters:T1WI (TR/TE:633/20ms, NEX:2), FsT2WI (TR/TE=5000/65ms, NEX:2), T2WI (TR/TE=6860/62ms, NEX:2). T2*WI (TR/TE=450/12ms, FA=20°, NEX:2). Coronal and sagittal scan, FOV=160mm,slice thickness=1.5nm, spacing=0mm.
2.Enhanced (1) Reactive hyperplasia lymph node group:the reactive hyperplasia lymph node groups of New Zealand white rabbits were divided into the SPIO enhanced group I (n=6), and anti-MECA-79-PBCA-USPIO group enhanced group Ⅱ (n=6).randomly. After the pre-scan the Group I the inject a dose of20umol Fe SPIO and the Group II injection a dose of20umol Fe anti-MECA-79-PBCA-USPIO for the12hours then scan as the same scan parameters;
The metastasis lymph node group model:the metastasis lymph node group of New Zealand white rabbits were divided into the SPIO enhanced group Ⅰ (n=6) and anti-MECA-79-PBCA-USPIO group enhanced group Ⅱ (n=6).randomly. After the pre-scan the Group I the inject a dose of20umol Fe SPIO and the Group II injection a dose of20umol Fe anti-MECA-79-PBCA-USPIO for the12hours then scan as the same scan parameters;
3、MRI imaging analysis
3.1The measurement of lymph node size
Measuring the size of lymph nodes in the MRI post-processing workstation on T1WI, The long and short diameter was measured presented as mean±standard deviation,
3.2Pre-scan and enhanced be measure the signal to noise ratio of the lymph nodes
The lymph node signal were measured in the different sequences on the pre-scan, the popliteal lymph nodes in the enhanced signal strength, calculated in each group the lymph nodes of the signal-to-noise ratio (Signal to noise ratio SNR), Evaluation SPIO and anti MECA-79-PBCA-USPIO in differentiating benign from malignant lymph nodes.
3.2Histopathological examination
All experimental animals in the enhanced MRI scan is completed, sacrificed the animal by air in the case of anesthesia, carefully expose popliteal lymph nodes and then remove and measure the short diameter of the lymph node, and calculating the number of lymph nodes. Then with10%neutral formalin soaked, fixed, paraffin-embedded, HE staining and Prussian blue staining to observe the popliteal fossa lymph node reactive hyperplasia and tumor infiltration, histopathology and MRI image in contrast.
Result
1. orthogonal experimental factorial analysis and analysis results showed that the optimum synthesis process for the anti-MECA-79-PBCA-USPIO nanoparticles was dextran-70concentration15%,the BCA concentration3%, pH value6.0.
2. Malvern laser particle size analyzer showed that PBCA-USPIO and anti-MECA-79-PBCA-USPIO's particle size and polydispersity were90nm and0.20,96nm and0.34nm.
3. Transmission electron microscope showed that the PBCA-USPIO, anti-MECA-79-PBCA-USPIO nanoparticles with uniform shape and size, spherical, shell-nuclear structure, the measurement of PBCA-USPIO, anti-MECA-79-PBCA-USPIO particle size approximately25±5nm,30±5nm.
4. The characteristic peaks of PBCA-USPIO and anti-MECA-79-PBCA-USPIO nanoparticles in X-ray diffraction the same to the standards X-ray of Fe3O4crystal Spectrum, It proved that the Fe3O4polymorphs of PBCA-USPIO and anti-MECA-79-PBCA-USPIO nanoparticles has no changed by parcels and reaction.
5. Anti-MECA-79-PBCA-USPIO has superparamagnetic.Derived from the hysteresis curves, the anti-MECA-79-PBCA-USPIO quality saturation magnetization were22emu/g Fe.
6. Anti-MECA-79-PBCA-USPIO nanoparticles relaxation rate results show that anti-MECA-79-PBCA-USPIO T2relaxation rate is0.159×106mol-1s-1.
7.1n vitro cytotoxicity experiments show that the anti-MECA-79-PBCA-USPIO has low toxicity to cells, when it reaches1000ug/ml, the cell survival rate is about70%, indicating that it has good biocompatibility and safety.
8. MRI pre-scan with different sequences was difficult to identify reactive hyperplasia lymp node from malignant metastatic lymph nodes;
9. The prepared anti-MECA79-PBCA-SPIO could targeted lymph nodes similar to SPIO and can be well distributed in the lymph nodes;
10. Anti-MECA79-PBCA-SPIO can diagnosis the reactive hyperplasia lymph nodes from metastatic lymph nodes with the correct diagnosis rate of about91.7%, which higher than the SPIO group of diagnostic accuracy rate about83.3%.
Conclusions
1, With the result of the orthogonal analysis, it confirm the best conditions for the preparation of PBCA-USPIO nanoparticles, and to make sure that the the PBCA-USPIO synthesis of the nanoparticle size, the PBCA-USPIO particle size synthetic uniform, circular shape or round, surface finished, liquid dispersion of the electron microscope particle size approximately25-30nm, water particle size of about90nm, X-Ray diffraction show Fe3O4crystal structure.
2,By streptavidin-biotin system successfully Synthesis anti-MECA-79-PBCA-USPIO, it's superparamagnetic T2magnetization saturation about22emu/g. The X-Ray diffraction show that Fe3O4crystals did not change after wrapped synthesis, anti-MECA-79-USPIO-PBCA fluid particle size is96nm, the T2relaxivity is0.159×106mol-1s-1.
3,The cytotoxicity effect of anti-MECA-79-PBCA-USPIO nanoparticles is small,show it is safe and biocompatible.
4. MRI pre-scan with different sequences was difficult to identify reactive hyperplasia lymp node from malignant metastatic lymph nodes;
5. Anti-MECA79-PBCA-SPIO can diagnosis reactive hyperplasia lymph nodes from metastatic lymph nodes with the correct diagnosis rate of about91.7%, which higher than the SPIO group of diagnostic accuracy rate about83.3%.
引文
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