新型MR叶酸靶向对比剂SPIO-PBCA纳米微粒的合成及实验室研究
|
摘要
研究目的
一、尝试在中性条件下制备叶酸靶向对比剂SPIO聚氰基丙烯酸正丁酯纳米微粒(SPIO-PBCA-NP),并获得成功；同时制备了非靶向的SPIO-PBCA-NP,以备后续实验作为对比；
二、在细胞实验中,将靶向与非靶向对比剂分别与人宫颈癌细胞Hela、将靶向对比剂与人肺腺癌细胞A549共孵育,考察SPIO-PBCA-FA-NP的细胞靶向效果,并探讨其潜在的应用价值；
三、在动物实验中,静脉注射SPIO-PBCA-NP,靶向实验组、非靶向对照组及阴性对照组的成像效果进行比较,考察SPIO-PBCA-FA-NP的肿瘤靶向强化效果,并探讨其潜在的应用价值。
研究方法
一、制备及表征
1、FA-PEG-NH2的合成与检验
将叶酸与PEG-(NH2)2按摩尔比1：1混合,13.2g叶酸溶于1mL DMSO,搅拌下加入一定量的DCC和NHS,加入120g PEG-(NH2)2,室温反应4h。加入5mL水,过滤除去不溶物,上清液冷冻干燥,乙醚洗涤干燥物,得到浅黄色固体FA-PEG-NH2。红外分光光度法检测所得固体产物。
2、靶向空白载体FA-PEG-PBCA的合成及形态观察
参考文献,先将PBCA单体用二氯甲烷稀释(VBCA:VCH2CL2=1:5),称取2.0g合成的FA-PEG-NH2溶于20ml去离子水中,用5ml注射器抽取2.7ml稀释后的BCA(浓度为1.5%v／v),在机械搅拌下逐滴加入,乳化聚合4小时,减压抽滤,既得FA-PEG-PBCA的胶体溶液。随后用0.45gm微孔滤膜过滤,将滤液存于4℃冰箱内备用。
1：4蒸馏水稀释纯净FA-PEG-PBCA-NP液体,取1滴放在镀膜铜网上,滤纸吸去多余液体,加入2%磷钨酸染30s,干燥后透射电镜下观察。
3、FA-PEG-PBCA合成验证试验
红外分光光度法检测冻干后的靶向空白载体及非靶向空白载体,比较二者及前者与FA-PEG-NH2的红外光谱图。
4、非靶向空白纳米微粒PBCA的制备及表面亲水修饰
同2,先将PBCA单体用二氯甲烷稀释(VBCA:VCH2CL2=1:5),称取2.0gNH2-PEG-NH2 (MW=4000Da)溶于20ml去离子水中,用5ml注射器抽取2.7ml稀释后的BCA(浓度为1.5%v／v),在机械搅拌下逐滴加入,乳化聚合4小时,减压抽滤,既得PEG亲水修饰PBCA后的胶体溶液。随后用0.45μm微孔滤膜过滤,将滤液存于4℃冰箱内备用。
5、靶向对比剂FA-PEG-PBCA-SPIO及非靶向对比剂PEG-PBCA-SPIO的合成及马尔文检测
首先将10mlSPIO加入2.7ml稀释后的BCA (VBCA:VCH2CL2=1:5,浓度为1.5%v／v),用5ml注射器抽取,在机械搅拌下逐滴加入至20ml含有2.0gFA-PEG-NH2 (MW=4000Da)的离子水中,乳化聚合4小时,减压抽滤。随后用0.45μm微孔滤膜过滤,将滤液存于4℃冰箱内备用。
同法,将10mlSPIO溶于2.7ml稀释后的BCA (VBCA:VCH2CL2=1:5,浓度为1.5%v／v),用5ml注射器抽取,在机械搅拌下逐滴加入至20ml含有2.0gNH2-PEG-NH2 (MW=4000Da)的去离子水中,乳化聚合4小时,减压抽滤。随后用0.45μm微孔滤膜过滤,将滤液存于4℃冰箱内备用。
粒径及其分布的测定：用Malvern-3000HS激光粒度分析仪测定粒径及其分布。吸取5μl制备的相应样品,用双蒸水稀释至量杯中。所选激光光源波长为633.0nm,测试温度为25.00±0.05。测得靶向对比剂平均粒径202.2nm,粒径分布为0.254；非靶向对比剂平均粒径210.7nm,粒径分布为0.336。6、靶向对比剂的电镜检测
(1)扫描电镜：首先,取少量透析后样品进行冷冻干燥呈粉末状。在扫描电镜的载物盘上粘上双面胶带,然后取少量粉末在胶带上靠近载物盘圆心部位,然后用吹气橡胶球朝载物盘径向朝外方向轻吹(注意不可用嘴吹气,以免唾液粘在样品上,也不可用工具拨粉末,以免破坏式样表面形貌),以使粉末可以均匀分布在胶带上,也可以把粘结不牢的粉末吹走(以免污染镜体)。然后在胶带边缘涂上导电银浆以连接样品与载物盘,等银浆干后将载物盘放入离子溅射仪中待真空度达到0.2Torr以下时,打开计时器(设定蒸金时间为5min),调节电流保持在4mA左右,这样就可以在样品表面均匀蒸涂一层足有20nm厚的金膜。然后将载物盘放入样品架即可进行观察。
(2)透射电镜：1：4稀释纯净的SPIO及PEG-PBCA-NP液体,各取1滴放在镀膜铜网上,滤纸吸去多余液体,加入2%磷钨酸染30s,充分干燥后透射电镜下观察。
7、包封率及载药量的测定
取5ml制备的未透析的SPIO-PBCA-NP,在12000rpm速度下低温离心30min,分别收集上清液及沉淀,用邻二氮菲法测定上清液的Fe浓度及自制的SPIO的Fe浓度,沉淀溶于水后按2方法透析,除去游离PEG,按公式计算包封率(ER)、载药量(DR)。
8、靶向对比剂T2时间测定
取5支试管,管径1.0cm,每管分别装浓度为0.1,0.12,0.14,0.16和0.18mmol/L的靶向对比剂10ml,另外取10ml纯净水及空白纳米粒溶液作对照。采用T2map(运用反转恢复快速自旋回波技术)序列对各管进行横断成像,8个回波,TE分别为20ms、40ms、60 ms、80ms、100ms、120ms、140ms、160ms, TR为2000ms,NEX为4,FOV为75×75cm,层厚2mm,层间隔2mm。T2 mapping原始数据使用Functool软件包的T2 mapping分析软件,可直接测T2值。
二、细胞试验
1、肿瘤细胞的培养及形态观察
选用表面叶酸受体阳性表达的人宫颈癌细胞株Hela与叶酸受体阴性表达的肺腺癌细胞株A549,培养条件采用含100μg/ml小牛血清的RPMI1640培养基,在37℃、5%CO2及饱和湿度的条件下连续培养及传代,使细胞处于贴壁状态。使用前,将处于对数生长期的该细胞用无叶酸RPMI1640培养基洗涤3次,并用该培养基继续培养1周。使用当天,再用不含血清的无叶酸RPMI1640培养基洗涤1次,并收集细胞、计数,调整细胞悬液的浓度至所需的浓度,备用。
对两种细胞分别进行HE染色及普鲁士蓝染色,观察细胞的形态。
2、细胞吞噬试验
将叶酸靶向及非靶向纳米微粒与宫颈癌细胞Hela在无叶酸RPMI1640培养基中共孵育,将叶酸靶向纳米微粒与肺腺癌细胞A549共孵育2小时后,利用普鲁士蓝染色观察细胞内含铁的情况,检测细胞对纳米微粒的靶向吸收情况。
3、叶酸竞争试验
将Hela细胞首先与游离叶酸共孵育30min,再与Fe浓度为100μg/ml的靶向对比剂共孵育2h,最后行普鲁士蓝染色。
三、SPIO-PBCA-NP的对荷瘤裸鼠的靶向性成像研究
1、肿瘤细胞的培养
选用表面叶酸受体阳性表达的人宫颈癌细胞株Hela及叶酸受体阴性表达的肺腺癌细胞株A549。培养条件采用含100μg/ml小牛血清及无叶酸RPMI1640培养基,在37℃、5%CO2及饱和温湿度的条件下传代培养,使细胞处于贴壁状态。
2、裸鼠荷人宫颈癌细胞及肺腺癌细胞皮下移植瘤动物模型的建立
BALB/C-nu/nu裸小鼠共18只,购自南方医科大学实验动物中心,鼠龄4-6周,体重18-24g,雌性。
裸小鼠饲养于南方医科大学动物中心符合SPF条件的裸鼠室。
裸鼠荷人宫颈癌及肺腺癌皮下移植瘤模型建立的步骤如下：
(1)将人宫颈癌Hela细胞株制成2×105／ml细胞悬液,用带7号针头的注射器抽取0.2ml,酒精棉球消毒后,选择裸小鼠腋下或大腿根部靠外侧皮下进行注射；
(2)裸鼠在标准超净环境下饲养约4-6周,待皮下移植瘤直径为1～1.5cm时开始后续试验；
(3)所有裸小鼠在进行后续实验前1周停止正常饮食,给予无叶酸食物。
3、实验动物分组及处理
将18只已接种人宫颈癌及肺腺癌皮下移植瘤的裸小鼠随机分成3组,每组6只：
(1)靶向实验组：接种人宫颈癌细胞Hela的裸小鼠,经鼠尾静脉注入叶酸靶向对比剂,注射剂量为5mgFe/kg;
(2)非靶向对照组：接种人宫颈癌细胞Hela的裸小鼠,经鼠尾静脉注入非靶向对比剂,注射剂量为5mgFe/kg;
(3)阴性对照组：接种人肺腺癌细胞A549的裸小鼠,经鼠尾静脉注入叶酸靶向对比剂,注射剂量为5mgFe/kg。
4、裸鼠MRI扫描：
利用SPIO的T2负性对比增强效应,通过测量T2WI上MRI信号强度,反映局部肿瘤组织含Fe的情况。
扫描仪器为GE公司的Signa Excite 3.0T MR成像系统,肘部线圈,行横断面及冠状面SE T2WI和T1WI扫描。SE T2WI采用快速自选回波(TSE)序列,TR/TE=4000/107.2,层厚2.0mm,层间距0.5mm,矩阵256×256,视野(Field of view, FOV) 75～100mm。SE T1WI扫描参数为TR/TE=600/16,层厚2.0mm,层间距0.5mm,矩阵256×256,FOV 75～100mm。
裸小鼠MRI检查步骤如下：
(1)以4.3%水合氯醛(430mg/kg)对裸小鼠进行腹腔注射麻醉；
(2)每只裸小鼠均先行平扫(plain scan),在注射对比剂前扫描；
(3)按分组要求,经鼠尾静脉分别注入叶酸靶向对比剂及非靶向对比剂,注射剂量为5mgFe/kg;
(4)注射对比剂后进行不同时间点的MRI扫描。扫描时间点选择为注射后后Oh(指注射后10min之内)、1h、4h、8h及24h,共5个不同的扫描时间点；
(5)MRI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。
5、MRI图像分析
测量增强前及增强后三组实验动物各时间点T2WI像及T1WI像上肿瘤、肌肉信号强度,在背景区域选取较大的ROI,测背景噪声的标准差(Standard deviation of the noise, SDN)。计算肿瘤及肌肉各时间点的信噪比(Signal to noise ratio, SNR)。
6、统计学分析
用SPSS 16.0软件包,采用重复测量的方差分析对比增强前后各组肿瘤及肌肉SNR是否有显著性差异。用单因素方差分析组间不同时间点肿瘤及肌肉SNR是否有显著性差异。P<0.05认为有显著性差异。
7、病理学检查
MRI检查完成后,迅速处死动物,切除肿瘤,观察肿瘤生长、浸润情况。用4%中性甲醛固定液固定标本、用石蜡包埋,连续切片,片厚1.5μm。每个组织块各取3套切片,分别行苏木素-伊红染色(Hematoxylin-Eosin, HE),叶酸受体的免疫组化检测和普鲁士蓝染色(Prussion Blue Staining),分别观察肿瘤组织常规光镜下的形态、叶酸受体的表达情况及组织内含Fe的情况。
结果
一、通过红外及透射电镜、扫描电镜检测,成功制备空白的叶酸靶向及非靶向PBCA-NP,其外观呈类圆形,大小欠均匀。
二、加入脂溶性SPIO后,电镜下可见靶向及非靶向对比剂具有明显的壳-核结构。
三、激光粒度分析仪测定靶向对比剂平均粒径为202.2nm,粒径分布为0.254；非靶向对比剂平均粒径为210.7nm,粒径分布为0.336。
四、制得的靶向对比剂含铁量为2.01mgFe/ml,包封率为76.91%,载药量为7.04%；非靶向对比剂含铁量为2.18mgFe/ml,包封率为83.42%,载药量7.63%；SPIO的含铁量为7.84mg/ml。
五、叶酸靶向对比剂与Hela细胞共孵育2小时后,普鲁士蓝染色细胞内可见不同量的蓝色铁颗粒；非叶酸靶向对比剂与Hela细胞、叶酸靶向对比剂与A549细胞共孵育2小时后,普鲁士蓝染色细胞内蓝染的铁颗粒含量极低。
六、叶酸竞争试验普鲁士蓝染色显示,Hela细胞内铁含量明显低于未与叶酸孵育后的Hela细胞；
七、荷人宫颈癌Hela裸鼠尾静脉分别注射靶向及非靶向对比剂进行MRI扫描显示,注射靶向对比剂后瘤体T2WI上信号降低明显；而非靶向对比剂降低不如靶向组明显；荷人肺腺癌A549裸鼠尾静脉注射靶向对比剂后,T2WI上瘤体信号减低不如靶向组明显。三组裸鼠注射对比剂后肌肉组织信号及T1WI上瘤体信号的改变不明显。
八、病理组织学检查,证实Hela细胞表面叶酸受体表达阳性,A549细胞表达阴性；靶向组裸鼠瘤体内含较多蓝染铁颗粒,非靶向组及阴性对照组瘤体内几乎不含铁颗粒。
结论
一、在中性条件下也可以成功制备PBCA-NP.
二.用接叶酸的PEG对PBCA-NP进行修饰,并包裹SPIO,得到叶酸靶向对比剂SPIO-PBCA-PEG-FA。
三、叶酸靶向SPIO-PBCA-NP对于表面表达叶酸受体的细胞具有良好的靶向性。
四、荷瘤裸鼠静脉注射叶酸靶向SPIO-PBCA-NP后,对于叶酸受体阳性的肿瘤具有良好的靶向性。
Objective
1. To prepare folate-targeted and non-targeted polybutylcyanoacrylate (PBCA) nanoparticles loaded with SPIO under neutral condition successfully;
2. To investigate the cell target effect of folate-targeted SPIO-PBCA-NP compared with folate-free SPIO-PBCA-NP in cell experiment with human cervical carcinoma Hela cell and human pulmonary adenocarcinoma A549 cell, and to explore its clinical potential.
3. To investigate the tumor target effect of SPIO-PBCA-FA-NP by intravenous injection of SPIO-PBCA-NP, compare the effect of imaging of folate-targeted SPIO-PBCA-NP, folate-free SPIO-PBCA-NP and negative control in animal experiment, and to explore its clinical potential.
Materials and Methods
Ⅰ. Preparation and characterization
1. Synthesis and analysis of FA-PEG-NH2 The folic acid and the PEG-(NH2)2 were mixed at the molar ratio of 1:1 as that 13.2g folic acid was dissolved in 1ml DMSO and a certain amount of DCC and NHS and 120g PEG-(NH2)2 was added under mechanical agitation. The reaction was carried out at room temperature for 4h. The 5 mL water was added and the insolubles were removed by filtration. The supernatant was free-dried and the Freeze-dry was washed for several times. At last, the yellow solid FA-PEG-NH2 was obtained which is detected by Infrared spectrophotometry.
2. Synthesis and morphology of the targeted blank carrier FA-PEG-PBCA
According to the references,2.7ml diluted BCA (1.5% v/v) was added in 20ml deionized water dissolving 2.0g synthetic FA-PEG-NH2 (MW=4000Da) with 5ml syringe in dropwise under mechanical agitation. The BCA was diluted by dichloromethane to 1:5. The polymerization process was carried out under mechanical agitation at room temperature for 4h. After vacuum filtration, the colloid solution of FA-PEG-PBCA was obtained which filtered with 0.45μm membrane, and stored in 4℃refrigerator.
A little of the diaylsed solution was dilluted with distilled water (1:4) and applied to metallic sample plate following negative staining with sodium phosphotungstate solution. The sample was dried absolutely and examined by transmission electron microscopy.
3. Analysis FA-PEG-PBCA with infrared spectrophotometry
The blank carrier FA-PEG-PBCA and the non-targeted blank carrier were detected by infrared spectrophotometry to compare the IR spectra between FA-PEG-PBCA and PEG-PBCA, FA-PEG-PBCA and FA-PEG-NH2.
4. Non-targeted blank PBCA-NP hydrophilic surface modification with PEG.
As above,2.7ml diluted BCA (1.5% v/v) was added in 20ml deionized water dissolving 2.0g NH2-PEG-NH2 (MW=4000Da) with 5ml syringe in dropwise under mechanical agitation. The BCA was diluted by dichloromethane to 1:5. The polymerization process was carried out under mechanical agitation at room temperature for 4h. After vacuum filtration, the colloid solution after PEG hydrophilic modified PBCA was obtained which filtered with 0.45μm membrane, and stored in 4℃refrigerator.
5. The synthesis and Malvern detection of targeted agent FA-PEG-PBCA-SPIO and non-targeted PEG-PBCA-SPIO
2.7ml diluted BCA (1.5% v/v) containing 10 ml SPIO was added in 20ml deionized water dissolving 2.0g FA-PEG-NH2 (MW=4000Da) with 5ml syringe in dropwise under mechanical agitation. The BCA was diluted by dichloromethane to 1:5. The polymerization process was carried out under mechanical agitation at room temperature for 4h. After vacuum filtration, the colloid solution after PEG hydrophilic modified PBCA was obtained which filtered with 0.45μm membrane, and stored in 4℃refrigerator.
As mentioned above,2.7ml diluted BCA (1.5% v/v) containing 10ml SPIO was added in 20ml deionized water dissolving 2.0g NH2-PEG-NH2 (MW=4000Da) with 5ml syringe in dropwise under mechanical agitation. The BCA was diluted by dichloromethane to 1:5. The polymerization process was carried out under mechanical agitation at room temperature for 4h. After vacuum filtration, the colloid solution after PEG hydrophilic modified PBCA was obtained which filtered with 0.45μm membrane, and stored in 4℃refrigerator.
NP size and size distribution measured:The NP size and size distribution were measured using dynamic light scattering (a Malvern Zetasizer 30000HS). A sample volume of 5μl was diluted with distilled water then measured under 633nm laser and a temperature of 25.00±0.05.
6. Electron microscope detection of targeted agents
SEM:A small amount of dialysis samples was freeze-dried into powder. The doubled-sided tape was fixed on the SEM's object plate and small amount of powder was put on the tape at the center of the plate. The powder was blown gently towards the radial outward direction of plate using inflatable rubber ball to make it uniformly distributed on the plate. And the rickety powder was also blown off to avoid contamination of the spectacle. (The mouth was forbidden to use so as not to blow saliva in the sample. Any tools were disabled to dial the powder to prevent damage to the morphology pattern). The conductive silver paste was overlaid on the tape edge in order to connect the sample with object plate. The plate was put into the ion sputtering instrument after the silver paste was dry. When the degree of vacuum was below 0.2Torr, the timer was opened (the steaming golden time was set to 5min) and the current was adjusted to around 4mA so that the sample can be uniformly coated with a surface layer of 20nm gold membrane. Then the object plate was put into the sample holder to observe.
TEM:A little SPIO and SPIO-PBCA-NP was dilluted with distilled water (1:4) and applied to metallic sample plate following negative staining with sodium phosphotungstate solution. The sample was dried absolutely and examined by transmission electron microscopy.
7. Measurement of entrapment rate and drug-loading rate
5ml undialysis SPIO-PBCA-NP was centrifugation at 1,2000 rpm for 30min and the supernatant and the precipitation were collected to measure entrapment rate and drug-loading rate, so as to the iron concentration of the domestic SPIO by Phenanthroline-spectrophotometry.
8. Measurement of the T2 time of the targeted agent
10ml purified water, blank nanoparticle and targeted contrast agent with the concentration of 0.1,0.12,0.14,0.16 and 0.18 mmol/L was put into seven test tubes (diameter 1.0cm). T2 map sequence with 8 echo was applied to get cross section images of the tubes (TE=20ms,40ms,60ms,80ms,100ms,120ms,140ms and 160ms, TR=2000ms, NEX=4, FOV=75×75cm, scanning thickness=2mm, interval=2mm), and T2 value of each tube could be measured through T2 mapping analysis software of Functool software package using the initial data of T2 mapping.
Ⅱ. Cellular experiment
1. Culture and morphological observation of tumor cells.
Human Cervical Carcinoma Line Hela and Human Lung Adenocarcinoma Line A549 was continuously cultured and passaging into RPMI1640 medium containing 100μg/ml bovine serum under condition of 37℃,5% CO2 and saturated humidity to keep them adherent. Before using, the cells in logarithmic phase was washed with folate-free RPMI1640 medium for 3 times, and cultured in folate-free RPMI1640 medium for 1 week. When using, the cells was washed with serum and folate-free RPMI1640 medium for one time, collected and counted. The concentration of cell suspension was adjusted to the desired levels to reserve.
Both types of cells undergo HE staining and Prussian Blue staining, and cell morphology are observed.
2. Phagocytosis test of tumor cell
Folate-targeted and non-targeted contrast agents were incubated with Human Cervical Carcinoma Line Hela and folate-targeted agent was incubated with Human Lung Adenocarcinoma Line A549 for 2 hours. Cellular iron incorporation and detention were observed by Prussian Blue staining to detect the iron absorption of the tumor cells.
3. Folic acid competitive examination
Free folic acid was incubated with Human Cervical Carcinoma Line Hela for 30min first, and then the targeted contrast agents with the Fe concentration being 100μg/ml for 2 hours. The intracellular iron was obserbed by Prussian blue staining.
Ⅲ. The study of SPIO-PBCA-NP targeted tumor-bearing nude mice imaging.
1. Culture of tumor cells
Human Cervical Carcinoma Line Hela and Human Lung Adenocarcinoma Line A549 was continuously cultured and passaging into RPMI1640 medium containing 100μg/ml bovine serum under condition of 37℃,5% CO2 and saturated humidity to keep them adherent.
2. Establishment of nude mice models bearing subcutaneously transplanted human cervical carcinoma and lung adenocarcinoma
18 BALB/C-nu/nu nude mice (Experimental Animal Center of Southern Medical University,4 to 6 weeks old, weighing 18～24g, female) were raised in the Southern Medical University Animal Center meeting the SPF (specific-pathgen free) conditions in the nude mouse room.
Steps of nude mice models bearing subcutaneously transplanted human cervical carcinoma and lung adenocarcinoma establishment were as follows:
(1) After disinfection,0.2ml Hela human cervical carcinoma cell suspension was put (2×105/ml) using syringe with 7# needle into the subaxillary subcutaneous tissue and the subcutaneous tissue of thgh of mice;
(2) The nude mice was raised in standard ultra-clean environment for 4 to 6 weeks until the diameter of implanted tumors reached 1～1.5cm;
(3) One week before the following experiment, all nude mice were given folate-free food instead of the normal diet.
3. Subgroup and treatment of experimental animal
Divide 18 nude mice bearing subcutaneously transplanted human cervical carcinoma and lung adenocarcinoma into three groups with 6 mice in each group
(1) Experimental group:The mice were injected folate-targeted contrast agent (5mg Fe/kg) into nude mice bearing Hela human cervical carcinoma through caudal vein;
(2) Non-targeted control group:The mice were injected nontargeted contrast agent (5mg Fe/kg) into nude mice bearing Hela human cervical carcinoma through caudal vein;
(3) Negative control group:The mice were inject folate-targeted contrast agent (5mg Fe/kg) into nude mice bearing A549 lung adenocarcinoma through caudal vein.
4. MRI scanning of the nude mice:
On basis of T2 negative reinforcement effect of SPIO, reflect the iron containing in tumor tissue was reflected by measuring the MRI signal intensity on T2WI.
All MR imagings were performed before and after different enhancement protocols at field strength of 3.0 Tesla MR scanner (Signa Excite, GE, USA) with use of a elbow coil for transmission and reception of the signal. MR sequences included T1-weighted spin echo images (repetition time [TR] mess/echo time [TE] mess, 600/16) and T2-weighted turbo spin echo images (TR/TE,4000/107.2). The thickness was 2.0mm, the space was 0.5mm, the matrix was 256 X 256, and FOVwas 75～100mm.
MRI Examination steps of nude mice were as follows:
(1) Nude mice were anesthetized by intraperitoneal injection of 4.3% chloral hydrate (430mg/kg);
(2) Each nude mice underwent a plain scan as baseline before enhancement;
(3) According to the grouping request, folate-targeted contrast agent (5mg Fe/kg) and non-targeted contrast agent (5mg Fe/kg) were intravenously injected in the nude mice by tail vein;
(4) Immediately after contrast medium injection, dynamic data of different group were aqcquired at 0h, 1h,4h,8h and 24h;
(5) When MRI scanning was completed, the data were transferred to post-processing workstation for the follow-up MRI image analysis.
5. MR image analysis
Average signal intensity (SI) over region-of-interest (ROI) drawn on tumors and muscles were measured on MR images. Background noise was measured in each image and its ROI was placed adjacent background outside mice. Signal to noise ratio (SNR) of tumors and muscles in each time points were calculated on T1WI imaging and T2WI in three groups.
6. Statistics analysis
SPSS 16.0 was used as analyzing software. Repetitive measurement ANOVA analyzed the difference in SNR of the tumors and muscles before and after enhancement. One-way ANOVA analyzed the difference in SNR of the tumors and muscles among different groups and time points. P>0.05 was regarded as no statistical difference; P<0.05 was regarded as statistical significant difference.
7. Pathology examination
After MR examination, the animals were put to death and the tumors were cut off to observe. Formalin-fixed, paraffin-embedded sections (1.5μm) were prepared. Two series slices were taken from each specimen to undergo Hematoxylin-Eosin staining, immunohistochemical staining and Prussion Blue Staining to observe the tumor appearance through light microscope, FR expression and iron content.
Results:
1. Folate-targeted and folate-free PBCA-NP were successfully prepared which was certifid by infrared, TEM and and SEM. It appeared as round-shaped uniform particle.
2. After adding liposoluble SPIO, folate-targeted and folate-free SPIO-PBCA-NP appeared as core-shell structure particle under TEM.
3. Measured by dynamic light scattering, the average size of targeted SPIO-PBCA-NP was 202.2nm and the index of size distribution was 0.254, the average size of folate-free SPIO-PBCA-NP was 210.7nm and the index of size distribution was 0.336.
4. The iron content of the targeted angent was 2.01mgFe/ml, entrapment rate was 76.91%, drug-loading rate was 7.04%; The iron content of the non-targeted angent was 2.18mgFe/ml, entrapment rate was 83.42%, drug-loading rate was 7.63%; The iron content of the domestic SPIO was 7.84mg/ml.
5. The Hela cells incubated with folate-targeted SPIO-PBCA-NP showed much higher intracellular iron density than the Hela cells with non-targeted agents and the A549 cells with folate-targeted SPIO-PBCA-NP.
6. Folic acid competitive examination and Prussian blue staining showed that intracellular iron density in Hela cells was significantly lower than non-folate incubated Hela cells.
7. On MRI, the signal intensity of tumors in nude mice with Hela human cervical carcinoma reduced significantly after intravenous injection of targeted contrast medium and lasted for a long time. The signal of tumors reduced insignificant compared with targeted group after intravenous injection of non-targeted agent. While the signal of the tumors in nude with human pulmonary adenocarcinoma A549 reduced insignificant after intravenous injection of targeted contrast medium. The signal of the muscles changed insignificant of these three group.
8. In histopathologic examination, the inmmunohistochemisty certified that Hela cells were positive expression of FR and A549 were negative expression; there were lots of blue iron particle in tumors of targeting gproup, while there were little in the non-targeting group and negative control group.
Conclusions:
1. PBCA-NP can be prepared successfully under neutral condition.
2. Folate-targeted SPIO-PBCA-PEG-FA can be prepared by means of surface modification PBCA-NP packed with SPIO using PEG linking falate.
3. SPIO-PBCA-FA-NP has good targeting tropism to the cells with folate receptor on their surface.
4. After intravenous injection, SPIO-PBCA-FA-NP has good targeting tropism to folate receptor-positive tumors in tumor-bearing mude mice.
一、尝试在中性条件下制备叶酸靶向对比剂SPIO聚氰基丙烯酸正丁酯纳米微粒(SPIO-PBCA-NP),并获得成功；同时制备了非靶向的SPIO-PBCA-NP,以备后续实验作为对比；
二、在细胞实验中,将靶向与非靶向对比剂分别与人宫颈癌细胞Hela、将靶向对比剂与人肺腺癌细胞A549共孵育,考察SPIO-PBCA-FA-NP的细胞靶向效果,并探讨其潜在的应用价值；
三、在动物实验中,静脉注射SPIO-PBCA-NP,靶向实验组、非靶向对照组及阴性对照组的成像效果进行比较,考察SPIO-PBCA-FA-NP的肿瘤靶向强化效果,并探讨其潜在的应用价值。
研究方法
一、制备及表征
1、FA-PEG-NH2的合成与检验
将叶酸与PEG-(NH2)2按摩尔比1：1混合,13.2g叶酸溶于1mL DMSO,搅拌下加入一定量的DCC和NHS,加入120g PEG-(NH2)2,室温反应4h。加入5mL水,过滤除去不溶物,上清液冷冻干燥,乙醚洗涤干燥物,得到浅黄色固体FA-PEG-NH2。红外分光光度法检测所得固体产物。
2、靶向空白载体FA-PEG-PBCA的合成及形态观察
参考文献,先将PBCA单体用二氯甲烷稀释(VBCA:VCH2CL2=1:5),称取2.0g合成的FA-PEG-NH2溶于20ml去离子水中,用5ml注射器抽取2.7ml稀释后的BCA(浓度为1.5%v／v),在机械搅拌下逐滴加入,乳化聚合4小时,减压抽滤,既得FA-PEG-PBCA的胶体溶液。随后用0.45gm微孔滤膜过滤,将滤液存于4℃冰箱内备用。
1：4蒸馏水稀释纯净FA-PEG-PBCA-NP液体,取1滴放在镀膜铜网上,滤纸吸去多余液体,加入2%磷钨酸染30s,干燥后透射电镜下观察。
3、FA-PEG-PBCA合成验证试验
红外分光光度法检测冻干后的靶向空白载体及非靶向空白载体,比较二者及前者与FA-PEG-NH2的红外光谱图。
4、非靶向空白纳米微粒PBCA的制备及表面亲水修饰
同2,先将PBCA单体用二氯甲烷稀释(VBCA:VCH2CL2=1:5),称取2.0gNH2-PEG-NH2 (MW=4000Da)溶于20ml去离子水中,用5ml注射器抽取2.7ml稀释后的BCA(浓度为1.5%v／v),在机械搅拌下逐滴加入,乳化聚合4小时,减压抽滤,既得PEG亲水修饰PBCA后的胶体溶液。随后用0.45μm微孔滤膜过滤,将滤液存于4℃冰箱内备用。
5、靶向对比剂FA-PEG-PBCA-SPIO及非靶向对比剂PEG-PBCA-SPIO的合成及马尔文检测
首先将10mlSPIO加入2.7ml稀释后的BCA (VBCA:VCH2CL2=1:5,浓度为1.5%v／v),用5ml注射器抽取,在机械搅拌下逐滴加入至20ml含有2.0gFA-PEG-NH2 (MW=4000Da)的离子水中,乳化聚合4小时,减压抽滤。随后用0.45μm微孔滤膜过滤,将滤液存于4℃冰箱内备用。
同法,将10mlSPIO溶于2.7ml稀释后的BCA (VBCA:VCH2CL2=1:5,浓度为1.5%v／v),用5ml注射器抽取,在机械搅拌下逐滴加入至20ml含有2.0gNH2-PEG-NH2 (MW=4000Da)的去离子水中,乳化聚合4小时,减压抽滤。随后用0.45μm微孔滤膜过滤,将滤液存于4℃冰箱内备用。
粒径及其分布的测定：用Malvern-3000HS激光粒度分析仪测定粒径及其分布。吸取5μl制备的相应样品,用双蒸水稀释至量杯中。所选激光光源波长为633.0nm,测试温度为25.00±0.05。测得靶向对比剂平均粒径202.2nm,粒径分布为0.254；非靶向对比剂平均粒径210.7nm,粒径分布为0.336。6、靶向对比剂的电镜检测
(1)扫描电镜：首先,取少量透析后样品进行冷冻干燥呈粉末状。在扫描电镜的载物盘上粘上双面胶带,然后取少量粉末在胶带上靠近载物盘圆心部位,然后用吹气橡胶球朝载物盘径向朝外方向轻吹(注意不可用嘴吹气,以免唾液粘在样品上,也不可用工具拨粉末,以免破坏式样表面形貌),以使粉末可以均匀分布在胶带上,也可以把粘结不牢的粉末吹走(以免污染镜体)。然后在胶带边缘涂上导电银浆以连接样品与载物盘,等银浆干后将载物盘放入离子溅射仪中待真空度达到0.2Torr以下时,打开计时器(设定蒸金时间为5min),调节电流保持在4mA左右,这样就可以在样品表面均匀蒸涂一层足有20nm厚的金膜。然后将载物盘放入样品架即可进行观察。
(2)透射电镜：1：4稀释纯净的SPIO及PEG-PBCA-NP液体,各取1滴放在镀膜铜网上,滤纸吸去多余液体,加入2%磷钨酸染30s,充分干燥后透射电镜下观察。
7、包封率及载药量的测定
取5ml制备的未透析的SPIO-PBCA-NP,在12000rpm速度下低温离心30min,分别收集上清液及沉淀,用邻二氮菲法测定上清液的Fe浓度及自制的SPIO的Fe浓度,沉淀溶于水后按2方法透析,除去游离PEG,按公式计算包封率(ER)、载药量(DR)。
8、靶向对比剂T2时间测定
取5支试管,管径1.0cm,每管分别装浓度为0.1,0.12,0.14,0.16和0.18mmol/L的靶向对比剂10ml,另外取10ml纯净水及空白纳米粒溶液作对照。采用T2map(运用反转恢复快速自旋回波技术)序列对各管进行横断成像,8个回波,TE分别为20ms、40ms、60 ms、80ms、100ms、120ms、140ms、160ms, TR为2000ms,NEX为4,FOV为75×75cm,层厚2mm,层间隔2mm。T2 mapping原始数据使用Functool软件包的T2 mapping分析软件,可直接测T2值。
二、细胞试验
1、肿瘤细胞的培养及形态观察
选用表面叶酸受体阳性表达的人宫颈癌细胞株Hela与叶酸受体阴性表达的肺腺癌细胞株A549,培养条件采用含100μg/ml小牛血清的RPMI1640培养基,在37℃、5%CO2及饱和湿度的条件下连续培养及传代,使细胞处于贴壁状态。使用前,将处于对数生长期的该细胞用无叶酸RPMI1640培养基洗涤3次,并用该培养基继续培养1周。使用当天,再用不含血清的无叶酸RPMI1640培养基洗涤1次,并收集细胞、计数,调整细胞悬液的浓度至所需的浓度,备用。
对两种细胞分别进行HE染色及普鲁士蓝染色,观察细胞的形态。
2、细胞吞噬试验
将叶酸靶向及非靶向纳米微粒与宫颈癌细胞Hela在无叶酸RPMI1640培养基中共孵育,将叶酸靶向纳米微粒与肺腺癌细胞A549共孵育2小时后,利用普鲁士蓝染色观察细胞内含铁的情况,检测细胞对纳米微粒的靶向吸收情况。
3、叶酸竞争试验
将Hela细胞首先与游离叶酸共孵育30min,再与Fe浓度为100μg/ml的靶向对比剂共孵育2h,最后行普鲁士蓝染色。
三、SPIO-PBCA-NP的对荷瘤裸鼠的靶向性成像研究
1、肿瘤细胞的培养
选用表面叶酸受体阳性表达的人宫颈癌细胞株Hela及叶酸受体阴性表达的肺腺癌细胞株A549。培养条件采用含100μg/ml小牛血清及无叶酸RPMI1640培养基,在37℃、5%CO2及饱和温湿度的条件下传代培养,使细胞处于贴壁状态。
2、裸鼠荷人宫颈癌细胞及肺腺癌细胞皮下移植瘤动物模型的建立
BALB/C-nu/nu裸小鼠共18只,购自南方医科大学实验动物中心,鼠龄4-6周,体重18-24g,雌性。
裸小鼠饲养于南方医科大学动物中心符合SPF条件的裸鼠室。
裸鼠荷人宫颈癌及肺腺癌皮下移植瘤模型建立的步骤如下：
(1)将人宫颈癌Hela细胞株制成2×105／ml细胞悬液,用带7号针头的注射器抽取0.2ml,酒精棉球消毒后,选择裸小鼠腋下或大腿根部靠外侧皮下进行注射；
(2)裸鼠在标准超净环境下饲养约4-6周,待皮下移植瘤直径为1～1.5cm时开始后续试验；
(3)所有裸小鼠在进行后续实验前1周停止正常饮食,给予无叶酸食物。
3、实验动物分组及处理
将18只已接种人宫颈癌及肺腺癌皮下移植瘤的裸小鼠随机分成3组,每组6只：
(1)靶向实验组：接种人宫颈癌细胞Hela的裸小鼠,经鼠尾静脉注入叶酸靶向对比剂,注射剂量为5mgFe/kg;
(2)非靶向对照组：接种人宫颈癌细胞Hela的裸小鼠,经鼠尾静脉注入非靶向对比剂,注射剂量为5mgFe/kg;
(3)阴性对照组：接种人肺腺癌细胞A549的裸小鼠,经鼠尾静脉注入叶酸靶向对比剂,注射剂量为5mgFe/kg。
4、裸鼠MRI扫描：
利用SPIO的T2负性对比增强效应,通过测量T2WI上MRI信号强度,反映局部肿瘤组织含Fe的情况。
扫描仪器为GE公司的Signa Excite 3.0T MR成像系统,肘部线圈,行横断面及冠状面SE T2WI和T1WI扫描。SE T2WI采用快速自选回波(TSE)序列,TR/TE=4000/107.2,层厚2.0mm,层间距0.5mm,矩阵256×256,视野(Field of view, FOV) 75～100mm。SE T1WI扫描参数为TR/TE=600/16,层厚2.0mm,层间距0.5mm,矩阵256×256,FOV 75～100mm。
裸小鼠MRI检查步骤如下：
(1)以4.3%水合氯醛(430mg/kg)对裸小鼠进行腹腔注射麻醉；
(2)每只裸小鼠均先行平扫(plain scan),在注射对比剂前扫描；
(3)按分组要求,经鼠尾静脉分别注入叶酸靶向对比剂及非靶向对比剂,注射剂量为5mgFe/kg;
(4)注射对比剂后进行不同时间点的MRI扫描。扫描时间点选择为注射后后Oh(指注射后10min之内)、1h、4h、8h及24h,共5个不同的扫描时间点；
(5)MRI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。
5、MRI图像分析
测量增强前及增强后三组实验动物各时间点T2WI像及T1WI像上肿瘤、肌肉信号强度,在背景区域选取较大的ROI,测背景噪声的标准差(Standard deviation of the noise, SDN)。计算肿瘤及肌肉各时间点的信噪比(Signal to noise ratio, SNR)。
6、统计学分析
用SPSS 16.0软件包,采用重复测量的方差分析对比增强前后各组肿瘤及肌肉SNR是否有显著性差异。用单因素方差分析组间不同时间点肿瘤及肌肉SNR是否有显著性差异。P<0.05认为有显著性差异。
7、病理学检查
MRI检查完成后,迅速处死动物,切除肿瘤,观察肿瘤生长、浸润情况。用4%中性甲醛固定液固定标本、用石蜡包埋,连续切片,片厚1.5μm。每个组织块各取3套切片,分别行苏木素-伊红染色(Hematoxylin-Eosin, HE),叶酸受体的免疫组化检测和普鲁士蓝染色(Prussion Blue Staining),分别观察肿瘤组织常规光镜下的形态、叶酸受体的表达情况及组织内含Fe的情况。
结果
一、通过红外及透射电镜、扫描电镜检测,成功制备空白的叶酸靶向及非靶向PBCA-NP,其外观呈类圆形,大小欠均匀。
二、加入脂溶性SPIO后,电镜下可见靶向及非靶向对比剂具有明显的壳-核结构。
三、激光粒度分析仪测定靶向对比剂平均粒径为202.2nm,粒径分布为0.254；非靶向对比剂平均粒径为210.7nm,粒径分布为0.336。
四、制得的靶向对比剂含铁量为2.01mgFe/ml,包封率为76.91%,载药量为7.04%；非靶向对比剂含铁量为2.18mgFe/ml,包封率为83.42%,载药量7.63%；SPIO的含铁量为7.84mg/ml。
五、叶酸靶向对比剂与Hela细胞共孵育2小时后,普鲁士蓝染色细胞内可见不同量的蓝色铁颗粒；非叶酸靶向对比剂与Hela细胞、叶酸靶向对比剂与A549细胞共孵育2小时后,普鲁士蓝染色细胞内蓝染的铁颗粒含量极低。
六、叶酸竞争试验普鲁士蓝染色显示,Hela细胞内铁含量明显低于未与叶酸孵育后的Hela细胞；
七、荷人宫颈癌Hela裸鼠尾静脉分别注射靶向及非靶向对比剂进行MRI扫描显示,注射靶向对比剂后瘤体T2WI上信号降低明显；而非靶向对比剂降低不如靶向组明显；荷人肺腺癌A549裸鼠尾静脉注射靶向对比剂后,T2WI上瘤体信号减低不如靶向组明显。三组裸鼠注射对比剂后肌肉组织信号及T1WI上瘤体信号的改变不明显。
八、病理组织学检查,证实Hela细胞表面叶酸受体表达阳性,A549细胞表达阴性；靶向组裸鼠瘤体内含较多蓝染铁颗粒,非靶向组及阴性对照组瘤体内几乎不含铁颗粒。
结论
一、在中性条件下也可以成功制备PBCA-NP.
二.用接叶酸的PEG对PBCA-NP进行修饰,并包裹SPIO,得到叶酸靶向对比剂SPIO-PBCA-PEG-FA。
三、叶酸靶向SPIO-PBCA-NP对于表面表达叶酸受体的细胞具有良好的靶向性。
四、荷瘤裸鼠静脉注射叶酸靶向SPIO-PBCA-NP后,对于叶酸受体阳性的肿瘤具有良好的靶向性。
Objective
1. To prepare folate-targeted and non-targeted polybutylcyanoacrylate (PBCA) nanoparticles loaded with SPIO under neutral condition successfully;
2. To investigate the cell target effect of folate-targeted SPIO-PBCA-NP compared with folate-free SPIO-PBCA-NP in cell experiment with human cervical carcinoma Hela cell and human pulmonary adenocarcinoma A549 cell, and to explore its clinical potential.
3. To investigate the tumor target effect of SPIO-PBCA-FA-NP by intravenous injection of SPIO-PBCA-NP, compare the effect of imaging of folate-targeted SPIO-PBCA-NP, folate-free SPIO-PBCA-NP and negative control in animal experiment, and to explore its clinical potential.
Materials and Methods
Ⅰ. Preparation and characterization
1. Synthesis and analysis of FA-PEG-NH2 The folic acid and the PEG-(NH2)2 were mixed at the molar ratio of 1:1 as that 13.2g folic acid was dissolved in 1ml DMSO and a certain amount of DCC and NHS and 120g PEG-(NH2)2 was added under mechanical agitation. The reaction was carried out at room temperature for 4h. The 5 mL water was added and the insolubles were removed by filtration. The supernatant was free-dried and the Freeze-dry was washed for several times. At last, the yellow solid FA-PEG-NH2 was obtained which is detected by Infrared spectrophotometry.
2. Synthesis and morphology of the targeted blank carrier FA-PEG-PBCA
According to the references,2.7ml diluted BCA (1.5% v/v) was added in 20ml deionized water dissolving 2.0g synthetic FA-PEG-NH2 (MW=4000Da) with 5ml syringe in dropwise under mechanical agitation. The BCA was diluted by dichloromethane to 1:5. The polymerization process was carried out under mechanical agitation at room temperature for 4h. After vacuum filtration, the colloid solution of FA-PEG-PBCA was obtained which filtered with 0.45μm membrane, and stored in 4℃refrigerator.
A little of the diaylsed solution was dilluted with distilled water (1:4) and applied to metallic sample plate following negative staining with sodium phosphotungstate solution. The sample was dried absolutely and examined by transmission electron microscopy.
3. Analysis FA-PEG-PBCA with infrared spectrophotometry
The blank carrier FA-PEG-PBCA and the non-targeted blank carrier were detected by infrared spectrophotometry to compare the IR spectra between FA-PEG-PBCA and PEG-PBCA, FA-PEG-PBCA and FA-PEG-NH2.
4. Non-targeted blank PBCA-NP hydrophilic surface modification with PEG.
As above,2.7ml diluted BCA (1.5% v/v) was added in 20ml deionized water dissolving 2.0g NH2-PEG-NH2 (MW=4000Da) with 5ml syringe in dropwise under mechanical agitation. The BCA was diluted by dichloromethane to 1:5. The polymerization process was carried out under mechanical agitation at room temperature for 4h. After vacuum filtration, the colloid solution after PEG hydrophilic modified PBCA was obtained which filtered with 0.45μm membrane, and stored in 4℃refrigerator.
5. The synthesis and Malvern detection of targeted agent FA-PEG-PBCA-SPIO and non-targeted PEG-PBCA-SPIO
2.7ml diluted BCA (1.5% v/v) containing 10 ml SPIO was added in 20ml deionized water dissolving 2.0g FA-PEG-NH2 (MW=4000Da) with 5ml syringe in dropwise under mechanical agitation. The BCA was diluted by dichloromethane to 1:5. The polymerization process was carried out under mechanical agitation at room temperature for 4h. After vacuum filtration, the colloid solution after PEG hydrophilic modified PBCA was obtained which filtered with 0.45μm membrane, and stored in 4℃refrigerator.
As mentioned above,2.7ml diluted BCA (1.5% v/v) containing 10ml SPIO was added in 20ml deionized water dissolving 2.0g NH2-PEG-NH2 (MW=4000Da) with 5ml syringe in dropwise under mechanical agitation. The BCA was diluted by dichloromethane to 1:5. The polymerization process was carried out under mechanical agitation at room temperature for 4h. After vacuum filtration, the colloid solution after PEG hydrophilic modified PBCA was obtained which filtered with 0.45μm membrane, and stored in 4℃refrigerator.
NP size and size distribution measured:The NP size and size distribution were measured using dynamic light scattering (a Malvern Zetasizer 30000HS). A sample volume of 5μl was diluted with distilled water then measured under 633nm laser and a temperature of 25.00±0.05.
6. Electron microscope detection of targeted agents
SEM:A small amount of dialysis samples was freeze-dried into powder. The doubled-sided tape was fixed on the SEM's object plate and small amount of powder was put on the tape at the center of the plate. The powder was blown gently towards the radial outward direction of plate using inflatable rubber ball to make it uniformly distributed on the plate. And the rickety powder was also blown off to avoid contamination of the spectacle. (The mouth was forbidden to use so as not to blow saliva in the sample. Any tools were disabled to dial the powder to prevent damage to the morphology pattern). The conductive silver paste was overlaid on the tape edge in order to connect the sample with object plate. The plate was put into the ion sputtering instrument after the silver paste was dry. When the degree of vacuum was below 0.2Torr, the timer was opened (the steaming golden time was set to 5min) and the current was adjusted to around 4mA so that the sample can be uniformly coated with a surface layer of 20nm gold membrane. Then the object plate was put into the sample holder to observe.
TEM:A little SPIO and SPIO-PBCA-NP was dilluted with distilled water (1:4) and applied to metallic sample plate following negative staining with sodium phosphotungstate solution. The sample was dried absolutely and examined by transmission electron microscopy.
7. Measurement of entrapment rate and drug-loading rate
5ml undialysis SPIO-PBCA-NP was centrifugation at 1,2000 rpm for 30min and the supernatant and the precipitation were collected to measure entrapment rate and drug-loading rate, so as to the iron concentration of the domestic SPIO by Phenanthroline-spectrophotometry.
8. Measurement of the T2 time of the targeted agent
10ml purified water, blank nanoparticle and targeted contrast agent with the concentration of 0.1,0.12,0.14,0.16 and 0.18 mmol/L was put into seven test tubes (diameter 1.0cm). T2 map sequence with 8 echo was applied to get cross section images of the tubes (TE=20ms,40ms,60ms,80ms,100ms,120ms,140ms and 160ms, TR=2000ms, NEX=4, FOV=75×75cm, scanning thickness=2mm, interval=2mm), and T2 value of each tube could be measured through T2 mapping analysis software of Functool software package using the initial data of T2 mapping.
Ⅱ. Cellular experiment
1. Culture and morphological observation of tumor cells.
Human Cervical Carcinoma Line Hela and Human Lung Adenocarcinoma Line A549 was continuously cultured and passaging into RPMI1640 medium containing 100μg/ml bovine serum under condition of 37℃,5% CO2 and saturated humidity to keep them adherent. Before using, the cells in logarithmic phase was washed with folate-free RPMI1640 medium for 3 times, and cultured in folate-free RPMI1640 medium for 1 week. When using, the cells was washed with serum and folate-free RPMI1640 medium for one time, collected and counted. The concentration of cell suspension was adjusted to the desired levels to reserve.
Both types of cells undergo HE staining and Prussian Blue staining, and cell morphology are observed.
2. Phagocytosis test of tumor cell
Folate-targeted and non-targeted contrast agents were incubated with Human Cervical Carcinoma Line Hela and folate-targeted agent was incubated with Human Lung Adenocarcinoma Line A549 for 2 hours. Cellular iron incorporation and detention were observed by Prussian Blue staining to detect the iron absorption of the tumor cells.
3. Folic acid competitive examination
Free folic acid was incubated with Human Cervical Carcinoma Line Hela for 30min first, and then the targeted contrast agents with the Fe concentration being 100μg/ml for 2 hours. The intracellular iron was obserbed by Prussian blue staining.
Ⅲ. The study of SPIO-PBCA-NP targeted tumor-bearing nude mice imaging.
1. Culture of tumor cells
Human Cervical Carcinoma Line Hela and Human Lung Adenocarcinoma Line A549 was continuously cultured and passaging into RPMI1640 medium containing 100μg/ml bovine serum under condition of 37℃,5% CO2 and saturated humidity to keep them adherent.
2. Establishment of nude mice models bearing subcutaneously transplanted human cervical carcinoma and lung adenocarcinoma
18 BALB/C-nu/nu nude mice (Experimental Animal Center of Southern Medical University,4 to 6 weeks old, weighing 18～24g, female) were raised in the Southern Medical University Animal Center meeting the SPF (specific-pathgen free) conditions in the nude mouse room.
Steps of nude mice models bearing subcutaneously transplanted human cervical carcinoma and lung adenocarcinoma establishment were as follows:
(1) After disinfection,0.2ml Hela human cervical carcinoma cell suspension was put (2×105/ml) using syringe with 7# needle into the subaxillary subcutaneous tissue and the subcutaneous tissue of thgh of mice;
(2) The nude mice was raised in standard ultra-clean environment for 4 to 6 weeks until the diameter of implanted tumors reached 1～1.5cm;
(3) One week before the following experiment, all nude mice were given folate-free food instead of the normal diet.
3. Subgroup and treatment of experimental animal
Divide 18 nude mice bearing subcutaneously transplanted human cervical carcinoma and lung adenocarcinoma into three groups with 6 mice in each group
(1) Experimental group:The mice were injected folate-targeted contrast agent (5mg Fe/kg) into nude mice bearing Hela human cervical carcinoma through caudal vein;
(2) Non-targeted control group:The mice were injected nontargeted contrast agent (5mg Fe/kg) into nude mice bearing Hela human cervical carcinoma through caudal vein;
(3) Negative control group:The mice were inject folate-targeted contrast agent (5mg Fe/kg) into nude mice bearing A549 lung adenocarcinoma through caudal vein.
4. MRI scanning of the nude mice:
On basis of T2 negative reinforcement effect of SPIO, reflect the iron containing in tumor tissue was reflected by measuring the MRI signal intensity on T2WI.
All MR imagings were performed before and after different enhancement protocols at field strength of 3.0 Tesla MR scanner (Signa Excite, GE, USA) with use of a elbow coil for transmission and reception of the signal. MR sequences included T1-weighted spin echo images (repetition time [TR] mess/echo time [TE] mess, 600/16) and T2-weighted turbo spin echo images (TR/TE,4000/107.2). The thickness was 2.0mm, the space was 0.5mm, the matrix was 256 X 256, and FOVwas 75～100mm.
MRI Examination steps of nude mice were as follows:
(1) Nude mice were anesthetized by intraperitoneal injection of 4.3% chloral hydrate (430mg/kg);
(2) Each nude mice underwent a plain scan as baseline before enhancement;
(3) According to the grouping request, folate-targeted contrast agent (5mg Fe/kg) and non-targeted contrast agent (5mg Fe/kg) were intravenously injected in the nude mice by tail vein;
(4) Immediately after contrast medium injection, dynamic data of different group were aqcquired at 0h, 1h,4h,8h and 24h;
(5) When MRI scanning was completed, the data were transferred to post-processing workstation for the follow-up MRI image analysis.
5. MR image analysis
Average signal intensity (SI) over region-of-interest (ROI) drawn on tumors and muscles were measured on MR images. Background noise was measured in each image and its ROI was placed adjacent background outside mice. Signal to noise ratio (SNR) of tumors and muscles in each time points were calculated on T1WI imaging and T2WI in three groups.
6. Statistics analysis
SPSS 16.0 was used as analyzing software. Repetitive measurement ANOVA analyzed the difference in SNR of the tumors and muscles before and after enhancement. One-way ANOVA analyzed the difference in SNR of the tumors and muscles among different groups and time points. P>0.05 was regarded as no statistical difference; P<0.05 was regarded as statistical significant difference.
7. Pathology examination
After MR examination, the animals were put to death and the tumors were cut off to observe. Formalin-fixed, paraffin-embedded sections (1.5μm) were prepared. Two series slices were taken from each specimen to undergo Hematoxylin-Eosin staining, immunohistochemical staining and Prussion Blue Staining to observe the tumor appearance through light microscope, FR expression and iron content.
Results:
1. Folate-targeted and folate-free PBCA-NP were successfully prepared which was certifid by infrared, TEM and and SEM. It appeared as round-shaped uniform particle.
2. After adding liposoluble SPIO, folate-targeted and folate-free SPIO-PBCA-NP appeared as core-shell structure particle under TEM.
3. Measured by dynamic light scattering, the average size of targeted SPIO-PBCA-NP was 202.2nm and the index of size distribution was 0.254, the average size of folate-free SPIO-PBCA-NP was 210.7nm and the index of size distribution was 0.336.
4. The iron content of the targeted angent was 2.01mgFe/ml, entrapment rate was 76.91%, drug-loading rate was 7.04%; The iron content of the non-targeted angent was 2.18mgFe/ml, entrapment rate was 83.42%, drug-loading rate was 7.63%; The iron content of the domestic SPIO was 7.84mg/ml.
5. The Hela cells incubated with folate-targeted SPIO-PBCA-NP showed much higher intracellular iron density than the Hela cells with non-targeted agents and the A549 cells with folate-targeted SPIO-PBCA-NP.
6. Folic acid competitive examination and Prussian blue staining showed that intracellular iron density in Hela cells was significantly lower than non-folate incubated Hela cells.
7. On MRI, the signal intensity of tumors in nude mice with Hela human cervical carcinoma reduced significantly after intravenous injection of targeted contrast medium and lasted for a long time. The signal of tumors reduced insignificant compared with targeted group after intravenous injection of non-targeted agent. While the signal of the tumors in nude with human pulmonary adenocarcinoma A549 reduced insignificant after intravenous injection of targeted contrast medium. The signal of the muscles changed insignificant of these three group.
8. In histopathologic examination, the inmmunohistochemisty certified that Hela cells were positive expression of FR and A549 were negative expression; there were lots of blue iron particle in tumors of targeting gproup, while there were little in the non-targeting group and negative control group.
Conclusions:
1. PBCA-NP can be prepared successfully under neutral condition.
2. Folate-targeted SPIO-PBCA-PEG-FA can be prepared by means of surface modification PBCA-NP packed with SPIO using PEG linking falate.
3. SPIO-PBCA-FA-NP has good targeting tropism to the cells with folate receptor on their surface.
4. After intravenous injection, SPIO-PBCA-FA-NP has good targeting tropism to folate receptor-positive tumors in tumor-bearing mude mice.
引文
[1]曾益新.肿瘤学,第二版.北京：人民卫生出版社,2003
[2]万德森.临床肿瘤学,第二版.北京：科学出版社,2000
[3]Hood JD, Bednarski M, Frausto R, et al. Tumor Regression by Targeted Gene Delivery to the Neovasculature. Science,2002,296:2404-7
[4]Langer R. Drug delivery and targeting. Nature,1998,3929suppl,n66790:5-12
[5]Langer R. New methods of drug delivery. Science,1990,249:1527-33
[6]Norased Nasongkla, Erik Bey, Jimin Ren, et al. Multifunctional Polymeric Micelles as Cancer-Targeted, MRI-Ultrasensitive Drug Delivery Systems. Nano letters.2006,6(11):2427-30
[7]Curtet C, Bourgoin C, Bohy J, et al. Gd-25DTPA-Mab, a potential NMR contrast agent for MRI in the xenografted nude mouse:preliminary studies. Int J Cancer Suppl,1998(supppl 2):126-32
[8]Anderson SA, Rader RK, Westlin WF, et al. Magnetic resonance contrast enhancement of neovasculature with alpha (v) beta (3)-targeted nanoparticles. Magn ResonMed,2000,44::434-9
[9]Jayson GC, Zweit J, Jackson A, et al. Molecular imaging and biological evaluation of HuMV833 anti-VEGF antibody:implications for trial design of antiangiogenic antibodies. J Natl Cancer Inst,2002,94:1484-93
[10]Konda SD, Aref M, Wang S, et al. Specific targeting of folate-dendrimer MRI contrast agents to the high affinity folate receptor expressed in ovarian tumor xenografts. MAGMA,2001,12(2):104-13
[11]滕皋军,以积极的姿态迎接分子影像学时代的来临.中华放射学杂志,2002,36(8)：679-80
[12]居胜红,陈锋,郑凯尔.MR分子影像学研究的进展.中华放射学杂志,2002,36(8)：747-50
[13]元建鹏,梁碧玲,钟镜联.MRI评价酪氨酸酶基因在人胚胎肾HEK293细胞表达的实验研究.中华放射学杂志,2004,38(6)：605-10
[14]周翠萍,沈君,梁碧玲.干细胞移植的磁性标记及磁共振成像活体示踪.国际放射医学核医学杂志,2006,30,(4)：253-6
[15]洪国斌,周经兴,沈君,等.叶酸受体介导的肿瘤靶向超顺磁性纳米胶束的 制备及体外实验.中华放射学杂志,2008,42(1)：19-23
[16]Guobin Hong, Renxu Yuan, Biling Liang, et al. Folate-funcionalized polymeric micelle as hepatic carcinoma-targeted, MRI-ultrasensitinve delivery system of antitumor drugs. Biomedical Microdevices,2008,10(5):693-700
[17]Hua Ai, Christopher Flask, Xintao Shuai, et al. Magnetite-loaded polymeric micelles as ultrasentitive magnetic-resonance probes. Advanced Materials. 2005,1:1949-52
[18]Hoon Choi, Seok, Rye Choi, et al. Iron oxide nanoparticles as magnetic resonance contrast agent for tumor imaging via folate receptor-targeted delivery. Acad Radio.2004,11(9):996-1004
[19]薛静,高培毅.超顺磁性氧化铁粒子标记神经干细胞与磁共振示踪成像研究进展.中华放射学杂志,2006,40(2)：213-5
[20]丁建辉,曾蒙苏,薛琼,等.叶酸受体型MR对比剂在荷人胃癌裸鼠诊断价值的实验研究.中华放射学杂志,2005,39(8)：882-6
[21]王劲武,黄其鎏,许乙凯等.钆标记抗人大肠癌单克隆抗体CL-3对裸鼠肿瘤模型MRI诊断的实验研究.中华放射学杂志,1998,32(6)：419-22
[22]Bhorade R, Weissleder R, Nokakoshi T, et al. Macrocyclic chelators with paramagnetic cations are internalized into mammalian cells via a HIV-tat derived membrane translocation peptide. Bioconjugate Chem,2000;11:301-5
[23]Bhujwalla ZM, Artemov D,Natarajan K, et al. Vascular differences detected by MRI for metastatic versus nonmetastatic breast and prostate cancer xenografts. Neoplasia.2001;3:143-53
[24]Weissleder R, Mahmood U. Molecular imaging. Radiology,2001; 219:316-33
[25]Weissleder R. A clearer vision for in vivo imaging. Nat Biotechnol, 2001,19(4):316-7
[26]Jaffer FA, Weissleder R. Molecular imaging in the clinical arena. JAMA, 2005,293:855-62
[27]Weissleder R, Mahmood U. Molecular imaging (review). Radiology, 2001,219(2):316-33
[28]Alencar H, Funovics MA, Figueiredo J, et al. Colonic adenocarcinomas: near-infrared microcatheter minging of smart probes for early detection:study in mice. Radiology,2007,244:232-8
[29]Sosnovik DE, Nahrendorf M, Deliolanis N, et al. Fluorescence tomography and magnetic resonance imaging of myocardial macrophage infiltration in infracted myocardium in vivo. Circulation,2007,115:1384-91
[30]Barrett T, Koyama Y, Hama Y, et al. In vivo diagnosis of epidermal growth factor receptor expression using molecular imaging with a cocktail of optically labeled monoclonal antibodies. Clin Cancer Res,2007,10:1078-119
[31]Webb B, Frame J, Zhao Z, Lee ML, et al. Molecular entrapment of small molecules within the interior of horse spleen ferritin. Arch Biochem Biophys. 1994;309:178-83
[32]Aime S, Geninatti Crich S, Frullano L. Compartmentalization of a gadolinium complex in the apoferritin cavity:a route to obtain high relaxivity contrast agents for magnetic resonance imaging. Angew Chem Int Ed Eng.2002;114:1059-61
[33]Wiener EC, Konda S, Shadron A, et al. Targeting dendrimer-chelates to tumors and tumor cells expressing the high affinity folate receptor. Invest Radiol. 1997;32:748-54
[34]Kabalka GW, Davis MA, Moss TH, et al. Gadolinium-labeled liposomes containing various amphiphilic Gd-DTPA derivatives:targeted MRI contrast enhancement agents for the liver. Magn Reson Med.1991;19:406-15
[35]Gohrrosenthal S, Scmittwillich H, Ebert W, et al. The demonstration of human tumors on nude mice using gadolinium labeled monoclonal antibodies for magnetic resonance imaging. Invest Radiol.1993;28:789-95
[36]Aime S, Botta M, Fedeli F, et al. High relaxivity contrast agents for magnetic resonance imaging based on multisite interactions between a beta-cyclodextrin oligomer and suitably functionalized Gd-Ⅲ chelates. Chem Eur J. 2001;7:5261-9
[37]E Allemann, R Gurny, E Doelker et al. Drug-loaded nanoparticles-preparation methods and drug targeting issues, Eur. J. Pharm.Biopharm.1993;39:173-91
[38]J Kreuter. Nanoparticulate systems for brain delivery of drugs, Adv. Drug Deliv. Rev.2001;47:65-81
[39]AC de Verdiere, C Dubernet, F Nemati, et al. Reversion of multidrug resistance with polyalkylcyanoacrylate nanoparticles:towards a mechanism of action, Br. J. Cancer.1997;76:198-205
[40]A.E. Gulyaev, S.E. Gelperina, I.N. Skidan,et al. Significant transport of doxorubicin into the brain with polysorbate 80-coated nanoparticles, Pharm. Res. 1999;16:1564-9
[41]CE. Soma, C Dubernet, D Bentolila, et al. Reversion of multidrug resistance by co-encapsulation of doxorubicin and cyclosporin A in polyalkylcyanoacrylate nanoparticles, Biomaterials.2000;21:1-7
[42]A Friese, E Seiller, G. Quack, et al. Increase of the duration of the anticonvulsive activity of a novel NMDA receptor an tagonist using poly(butylcyanoacrylate) nanoparticles as a parenteral controlled release system, Eur. J. Pharm. Biopharm. 2000;49:103-9
[43]Kataoka K, Harada A, Nagasaki Y. Block copolymer micelles for drug delivery: design, characterization and biological significance. Adv Drug Deliv Rev.2001; 47:113-31
[44]Torchilin VP, Levchenko TS, Whiteman KR, et al. Amphiphilic poly-N-vinylpyrrolidones:synthesis, properties and liposome surface modification. Biomaterials.2001; 22:3035-44
[45]Otsuka H, Nagasaki Y, Kataoka K et al. PEGylated nanoparticles for biological and pharmaceutical applications. Adv.Drug Deliv Rev.2003;55:403-19
[46]Leamon CP, Low PS. Folate-mediated targeting:from diagnositics to drug and gene delivery. Drug Discov Today,2001,6:44-51
[47]肖苏尧,童春义,刘选明,等.肿瘤靶向性药物载体叶酸-淀粉纳米颗粒的研制与应用.科学通报,2006,51(10)：1151-5
[48]Campbell IG, Jones TA, Foulkes WD, et al. Folate-binding protein is a marker for ovarian cancer, Cancer Res.1991,51:5329-38
[49]Lu Y, Low PS. Immunotherapy of folate receptor-expressing tumors:review of recent advances and future prospects (Review). J Control Release, 2003,91(1):17-29
[50]Bottero F, Tomassetti A, Canevari S, et al. Gene transfection and expression of the ovarian carcinoma marker folate binding protein on NIH/3T3 cells increases cell growth in vitro and in vivo. Cancer Res,1993,53:5791-6
[51]Holm J, Hansen SI, Hoier-Madsen M, et al. Folate receptor of human mammary adenocarcitime, noma. APMIS,1994,102:413-9
[52]Ross JF, Chaudhuri PK, Ratnam M, Differential regulation of folate receptor isoforms in normal and malignant tissuses in vivo and in established cell lines: physiologic and clinical implications. Cancer,1994,73:2431-43
[53]Gruner BA, Weitman SD. The folate receptor as a potential therapeutic anticancer target, Invest. New Drugs,1998,16:205-19
[54]陆伟跃,刘敏,潘俊,等.叶酸-脂质体制备及对Hela细胞靶向作用.上海医科大学学报,2000,27：4-8
[55]Gottschal S, Cristiano RJ, Smith LC, et al. Folate receptor mediated DNA delivery into tumor cells:potosomal disruption results in enhanced gene expression. Gene Ther,1994,1:185-91
[56]Reddy JA, Dean D, Kennedy MD, et al. Optimization of folate-conjugated liposomal vectors for folate receptor-mediated gene therapy. J Pharm Sci, 1999,88(11):1112-8
[57]Teruaki H, Tomohisa F, Shuichi H, et al. Schizophyllan-folate conjugate as a new non-cytotoxic and cancer-targeted antisense carrier. Bioorg Med Chem Lett, 2005,15:327-30
[1]任非,陈建海,陈志良.丝裂霉素C-聚氰基丙烯酸正丁酯磁性纳米球的制备方法及性质研究.中国药学杂志,2005,40(15)：1163-6
[2]任非,陈建海,杨西晓,等.丝裂霉素C聚氰基丙烯酸正丁酯磁性纳米球对肝细胞L-02的生长抑制作用.中国药理学通报,2004,20(10)：1126-30
[3]蔡林,胡治平,牛光峰,等.磁性阿霉素纳米药物研制及其靶向性的实验研究.中国骨肿瘤骨病,2008,7(4)：225-30
[4]Couvreur P, Kante B, Roland M, et al. Polycyanoacrylate nanocapsules as potential lysosomotropic carrier:Preparation, morphological and sorptive properties. J Pham Phamacol,1979,31:331-3
[5]张胜,胡蕴玉,吴道澄.骨形态发生蛋白纳米型缓释制剂的制备及性质.第四军医大学学报,2002,23(18)：1686-9
[6]张志荣,廖工铁.聚氰基丙烯酸酯昊微粒的研究进展.中国药学杂志,1994,29：323-6
[7]Vanthier C, Dubernet C, Fattal E, et al. Poly(alkycyanoacrylates) as biodegradable materials for biomedical applications. Advanced Drug Delivery Review,2003,55:519-48
[8]Schroeder U, Sabel BA, Schroeder H. Diffusion enhancement of drugs by loaded nanoparticles in vitro. Prog Neuropsychopharmacol Biol Psychiatry, 1999,23(5):941-9
[9]马淑燕,欧阳五庆,,刘玉梅,等.以聚氰基丙烯酸正丁酯为载体长春碱纳米粒的制备和性能评价及其对大鼠C6脑神经胶质瘤细胞生长的影响.中国组织工程研究与临床康复,2008,12(19)：3757-60
[10]桂凯,张阳德.载阿霉素聚氰基丙烯酸正丁酯纳米粒对L-02细胞的毒性研究.中国普通外科杂志,2008,17(2)：159-61
[11]Lenaerts V, Couvreur P, Christiaens-Lyyh D, et al. Degradation of poly (isobutyl cyanoncylate) nanoparticles. Biomaterials,1984,5(2):65-8
[12]Muller RH, Lherm C, Herbort J, et al. In vitro modal for the degradation of alkylcyanoacrylate nanoparticles. Biomaterials,1990,11(8):590-5
[13]Kreuter J, Wilson CG, Fry JR, et al. Toxicity and association of polycyanoacrylate nanoparticles with hepatocytes. J Microencapsul, 1984,1(3):253-7
[14]金武龙,周梁,郭慧玲,等.纳米微粒靶向治疗兔舌癌的实验研究.北京口腔医学,2008,16(4)：181-4
[15]Olivier JC. Drug transport to brain with targeted nanoparticles. NeuroRx, 2003,2(1),108-19
[16]SI Park, JH Lim, YH Hwang, et al. In vivo and in vitro antitumor activity of doxorubicin-loaded magnetic fluids. Phy Sstat Sol,2007,12(4):4345-51
[17]姜子荣,杨西晓,兰青.DOX-PBCA-NP的制备及其对脑胶质瘤SHG44细胞的抑制作用观察.山东医药,2008,48(12)：22-4
[18]Phelps ME.PET:The merging of biolog and imaging into molecular imaging. Nucl Med.2000; 41:661-81
[19]Artemov D. Molecula magnetic resonance imaging with targeted contrast agents. J Cell Bio chem.2003; 909:518-24
[20]Heath JR, Phelps ME, HoodL, etal. Nanosystems biology. Mol Imaging Biol. 2003;5:312-25
[21]何勤,张志荣.万乃洛韦聚氰基丙烯酸正丁酯毫微粒制备工艺的优选.华西药学杂志,1998,13(1)：14-6
[22]吕国士,许乙凯.新型MR特异性对比剂Gd-聚氰基丙烯酸正丁酯纳米微粒的合成及其肝脏靶向性作用.中国组织工程研究与临床康复,2008,12(1)：43-6
[23]Giacomo F, Mariano L, Silvana M. Amoxicillin-loaded polyethylcyanoacrylate nanoparticles:Influence of PEG coating on the particle size, drug release rate and phagocytic uptake. Biomaterials,2001,22:2857-65
[24]Gontana G, Potarresi G, Tomarchio V, et al. Preparation characterization and in vitro antimicrobial activity of ampicillin-loaded polyethylcyanoacrylate nanoparticles. Biomaterials,1998,19(11-12):1009
[25]Gregoriadis G. Liposome Technology (Vol 3). Boca Raton:CRC Press, 1984:75-94
[26]张兆旺.重要药剂学.北京：中国中医药出版社,2003：508
[27]Yu Zhang, Siyu Zhu, Lichen Yin, et al. Preparation, characterization and biocompatibility of poly (ethylene glyol)-poly(n-butyl cyanoacrylate) nanocapsules with oil core via miniemulsion polymerization. European Polymer Journal,2008,44:1654-61
[1]Weitman SD, Lark RH, Coney LR, et al. Distribution of the folate receptor GP38 in normal and malignant cell lines and tissuses. Cancer Res,1992,52:3396-401
[2]Lee RJ, Huang L. Folate-targeted, anionic liposome-enliposome-trapped polylysine-condensed DNA for tumor cell-specific gene transfer. J Biol Chem, 1996,271:8481-7
[3]Anderson GB. Folate-binding proteins. Ann Rev Nutr,1990,10:319-35
[4]谢俊玲,沈丹华,杜金荣.叶酸受体α在女性生殖系统肿瘤中的研究进展.中国妇产科临床杂志,2008.9(4)：312-5
[5]Kelemen LE. The role of folate receptor a in cancer development, progression and treatment cause, consequence or innocent bystander. Int J Cancer, 2006,119:243-50
[6]陈慧,李娟.受体介导的肿瘤靶向治疗研究进展.药学与临床研究, 2008,16(4)：288-92
[7]Antony AC. The biological chemistry of folate receptors. Blood, 1992,79(11):2807-20
[8]张寿春,李慧敏,王柯敏.叶酸-聚合物靶向载药系统研究进展.中国医药工业杂志,2007,38(9)：676-80
[9]邢诗安.叶酸受体及其在肿瘤核医学中的应用.国外医学放射医学核医学分册,2002,26(6)：257-9
[10]Konda SD, Aref M, Wang S, et al. Specific targeting of folate-dendrimer MRI contrast agents to the high affinity folate receptor expressed in ovarian tumor xenografts. MAGMA,2001,12(2):104-13
[11]Hoon Choi, Seok, Rye Choi, et al. Iron oxide nanoparticles as magnetic resonance contrast agent for tumor imaging via folate receptor-targeted delivery. Acad Radio.2004,11(9):996-1004
[12]王劲武,黄其鎏,许乙凯等.钆标记抗人大肠癌单克隆抗体CL-3对裸鼠肿瘤模型MRI诊断的实验研究.中华放射学杂志,1998,32(6)：419-22
[13]肖苏尧,童春义,刘选明,等.肿瘤靶向性药物载体叶酸-淀粉纳米颗粒的研制与应用.科学通报,2006,51(10)：1151-5
[14]Campbell IG, Jones TA, Foulkes WD, et al. Folate-binding protein is a marker for ovarian cancer, Cancer Res.1991,51:5329-38
[15]丁建辉,曾蒙苏,薛琼,等.叶酸受体型MR对比剂在荷人胃癌裸鼠诊断价值的实验研究.中华放射学杂志,2005,39(8)：882-6
[1]陈慧,李娟.受体介导的肿瘤靶向治疗研究进展.药学与临床研究,2008,16(4)：288-92
[2]张寿春,李慧敏,王柯敏.叶酸-聚合物靶向载药系统研究进展.中国医药工业杂志,2007,38(9)：676-80
[3]Ross JF, Chaudhuri PK, Ratnam M, Differential regulation of folate receptor isoforms in normal and malignant tissuses in vivo and in established cell lines: physiologic and clinical implications. Cancer,1994,73:2431-43
[4]Weitman SD, Lark RH, Coney LR, et al. Distribution of the folate receptor GP38 in normal and malignant cell lines and tissuses. Cancer Res,1992,52:3396-401
[5]高晓宁,唐锁勤.叶酸受体及其在介导药物靶向肿瘤细胞治疗中的应用.中国实验血液学杂志,2005,13(5)：911-4
[6]Weitman SD, Weinberg AG, Coney LR, et al. Cellular localization of the folate receptor:potential role in drug toxicity and folate homeostasis. Cancer Res, 1992,52:6708-11
[7]Bagnoli M, Canevari S, Figini M, et al. A step further in understanding the biology of the folate receptor in ovarian carcinomar. Gynecol Oncol, 2003,88:S140-4
[8]林凤云,朱照静.叶酸受体介导的靶向给药研究进展.中国医院药学杂志,2006,26(10)：1281-4
[9]Garin-Chesa P, Campbell I, Saigo PE, et al. Trophoblast and ovarian cancer antigen LK26. Sensitivity and specificity in immunapathology and molecular identification as a folate-binding protein. Am J Pathol,1993,142(2):557-67
[10]Cancer Facts and Figures 2007, American Cancer Society, Inc, Atlanta, Georgia, USA (www.cancer.org)
[11]Bueno R, Appasani K, Mercer H, et al. The alpha folate receptor is highly activated in malignant pleural mesothelioma. Thorac. Cardiovasc. Surg, 2001,121(2):225-33
[12]丁建辉,曾蒙苏,薛琼,等.叶酸受体型MR对比剂在荷人胃癌裸鼠诊断价值的实验研究.中华放射学杂志,2005,39(8)：882-6
[13]Bottero F, Tomassetti A, Canevari S, et al. Gene transfection and expression of the ovarian carcinoma marker folate binding protein on NIH/3T3 cells increases cell growth in vitro and in vivo. Cancer Res,1993,53:5791-6
[14]Holm J, Hansen SI, Hoier-Madsen M, et al. Folate receptor of human mammary adenocarcitime, noma. APMIS,1994,102:413-9
[15]Gruner BA, Weitman SD. The folate receptor as a potential therapeutic anticancer target, Invest. New Drugs,1998,16:205-19
[16]陆伟跃,刘敏,潘俊,等.叶酸-脂质体制备及对Hela细胞靶向作用.上海医科大学学报,2000,27：4-8
[17]Gottschal S, Cristiano RJ, Smith LC, et al. Folate receptor mediated DNA delivery into tumor cells:potosomal disruption results in enhanced gene expression. Gene Ther,1994,1:185-91
[18]Reddy JA, Dean D, Kennedy MD, et al. Optimization of folate-conjugated liposomal vectors for folate receptor-mediated gene therapy. J Pharm Sci, 1999,88(11):1112-8
[19]Teruaki H, Tomohisa F, Shuichi H, et al. Schizophyllan-folate conjugate as a new non-cytotoxic and cancer-targeted antisense carrier. Bioorg Med Chem Lett, 2005,15:327-30
[20]Leamon CP, Reddy JA. Folate-targeted chemotherapy. Adv Drug Deliv Rev, 2004,56(8):1127-41
[21]Konda SD, Aref M, Brechbiel M, et al. Development of a tumor-targeting MR contrast agent using the high-affinity folate receptor:work in progress. Invest Radiol,2000,35:50-7
[22]洪国斌,梁碧玲,沈君,等.三维梯度回波磁敏感成像在颅内出血诊断中的初步应用.实用放射学杂志,2008,24(2)：151-4
[23]Natha DW, Judy C, Barbara H, et al. Reliability in detection of hemorrhage in acute stroke by a new three-dimensional gradient recalled echo susceptibility-weighted imaging technique compared to computed tomography: A retrospective study, J Magn Reson Imagin,2994,20(2):372-7
[24]E Mark H, Xu YB, Cheng YC, et al. Susceptibility Weighted Imaging (SWI). Magn Reson Med,2004,52(4):612-8
[25]Wolff SD, Balaban RS. Magnetization transfer imaging:practical aspects and clinical applications. Radiology,1994,192:593-9
[26]Koh DM, Collins DJ. Diffusion-weighted MRI in the body:applications and challenges in oncology. AJR,2007,188(6):1622-35
[27]Gallbraith SM. MR in oncology drug development. NMR Biomed, 2006;19(6):681-9
[2]万德森.临床肿瘤学,第二版.北京：科学出版社,2000
[3]Hood JD, Bednarski M, Frausto R, et al. Tumor Regression by Targeted Gene Delivery to the Neovasculature. Science,2002,296:2404-7
[4]Langer R. Drug delivery and targeting. Nature,1998,3929suppl,n66790:5-12
[5]Langer R. New methods of drug delivery. Science,1990,249:1527-33
[6]Norased Nasongkla, Erik Bey, Jimin Ren, et al. Multifunctional Polymeric Micelles as Cancer-Targeted, MRI-Ultrasensitive Drug Delivery Systems. Nano letters.2006,6(11):2427-30
[7]Curtet C, Bourgoin C, Bohy J, et al. Gd-25DTPA-Mab, a potential NMR contrast agent for MRI in the xenografted nude mouse:preliminary studies. Int J Cancer Suppl,1998(supppl 2):126-32
[8]Anderson SA, Rader RK, Westlin WF, et al. Magnetic resonance contrast enhancement of neovasculature with alpha (v) beta (3)-targeted nanoparticles. Magn ResonMed,2000,44::434-9
[9]Jayson GC, Zweit J, Jackson A, et al. Molecular imaging and biological evaluation of HuMV833 anti-VEGF antibody:implications for trial design of antiangiogenic antibodies. J Natl Cancer Inst,2002,94:1484-93
[10]Konda SD, Aref M, Wang S, et al. Specific targeting of folate-dendrimer MRI contrast agents to the high affinity folate receptor expressed in ovarian tumor xenografts. MAGMA,2001,12(2):104-13
[11]滕皋军,以积极的姿态迎接分子影像学时代的来临.中华放射学杂志,2002,36(8)：679-80
[12]居胜红,陈锋,郑凯尔.MR分子影像学研究的进展.中华放射学杂志,2002,36(8)：747-50
[13]元建鹏,梁碧玲,钟镜联.MRI评价酪氨酸酶基因在人胚胎肾HEK293细胞表达的实验研究.中华放射学杂志,2004,38(6)：605-10
[14]周翠萍,沈君,梁碧玲.干细胞移植的磁性标记及磁共振成像活体示踪.国际放射医学核医学杂志,2006,30,(4)：253-6
[15]洪国斌,周经兴,沈君,等.叶酸受体介导的肿瘤靶向超顺磁性纳米胶束的 制备及体外实验.中华放射学杂志,2008,42(1)：19-23
[16]Guobin Hong, Renxu Yuan, Biling Liang, et al. Folate-funcionalized polymeric micelle as hepatic carcinoma-targeted, MRI-ultrasensitinve delivery system of antitumor drugs. Biomedical Microdevices,2008,10(5):693-700
[17]Hua Ai, Christopher Flask, Xintao Shuai, et al. Magnetite-loaded polymeric micelles as ultrasentitive magnetic-resonance probes. Advanced Materials. 2005,1:1949-52
[18]Hoon Choi, Seok, Rye Choi, et al. Iron oxide nanoparticles as magnetic resonance contrast agent for tumor imaging via folate receptor-targeted delivery. Acad Radio.2004,11(9):996-1004
[19]薛静,高培毅.超顺磁性氧化铁粒子标记神经干细胞与磁共振示踪成像研究进展.中华放射学杂志,2006,40(2)：213-5
[20]丁建辉,曾蒙苏,薛琼,等.叶酸受体型MR对比剂在荷人胃癌裸鼠诊断价值的实验研究.中华放射学杂志,2005,39(8)：882-6
[21]王劲武,黄其鎏,许乙凯等.钆标记抗人大肠癌单克隆抗体CL-3对裸鼠肿瘤模型MRI诊断的实验研究.中华放射学杂志,1998,32(6)：419-22
[22]Bhorade R, Weissleder R, Nokakoshi T, et al. Macrocyclic chelators with paramagnetic cations are internalized into mammalian cells via a HIV-tat derived membrane translocation peptide. Bioconjugate Chem,2000;11:301-5
[23]Bhujwalla ZM, Artemov D,Natarajan K, et al. Vascular differences detected by MRI for metastatic versus nonmetastatic breast and prostate cancer xenografts. Neoplasia.2001;3:143-53
[24]Weissleder R, Mahmood U. Molecular imaging. Radiology,2001; 219:316-33
[25]Weissleder R. A clearer vision for in vivo imaging. Nat Biotechnol, 2001,19(4):316-7
[26]Jaffer FA, Weissleder R. Molecular imaging in the clinical arena. JAMA, 2005,293:855-62
[27]Weissleder R, Mahmood U. Molecular imaging (review). Radiology, 2001,219(2):316-33
[28]Alencar H, Funovics MA, Figueiredo J, et al. Colonic adenocarcinomas: near-infrared microcatheter minging of smart probes for early detection:study in mice. Radiology,2007,244:232-8
[29]Sosnovik DE, Nahrendorf M, Deliolanis N, et al. Fluorescence tomography and magnetic resonance imaging of myocardial macrophage infiltration in infracted myocardium in vivo. Circulation,2007,115:1384-91
[30]Barrett T, Koyama Y, Hama Y, et al. In vivo diagnosis of epidermal growth factor receptor expression using molecular imaging with a cocktail of optically labeled monoclonal antibodies. Clin Cancer Res,2007,10:1078-119
[31]Webb B, Frame J, Zhao Z, Lee ML, et al. Molecular entrapment of small molecules within the interior of horse spleen ferritin. Arch Biochem Biophys. 1994;309:178-83
[32]Aime S, Geninatti Crich S, Frullano L. Compartmentalization of a gadolinium complex in the apoferritin cavity:a route to obtain high relaxivity contrast agents for magnetic resonance imaging. Angew Chem Int Ed Eng.2002;114:1059-61
[33]Wiener EC, Konda S, Shadron A, et al. Targeting dendrimer-chelates to tumors and tumor cells expressing the high affinity folate receptor. Invest Radiol. 1997;32:748-54
[34]Kabalka GW, Davis MA, Moss TH, et al. Gadolinium-labeled liposomes containing various amphiphilic Gd-DTPA derivatives:targeted MRI contrast enhancement agents for the liver. Magn Reson Med.1991;19:406-15
[35]Gohrrosenthal S, Scmittwillich H, Ebert W, et al. The demonstration of human tumors on nude mice using gadolinium labeled monoclonal antibodies for magnetic resonance imaging. Invest Radiol.1993;28:789-95
[36]Aime S, Botta M, Fedeli F, et al. High relaxivity contrast agents for magnetic resonance imaging based on multisite interactions between a beta-cyclodextrin oligomer and suitably functionalized Gd-Ⅲ chelates. Chem Eur J. 2001;7:5261-9
[37]E Allemann, R Gurny, E Doelker et al. Drug-loaded nanoparticles-preparation methods and drug targeting issues, Eur. J. Pharm.Biopharm.1993;39:173-91
[38]J Kreuter. Nanoparticulate systems for brain delivery of drugs, Adv. Drug Deliv. Rev.2001;47:65-81
[39]AC de Verdiere, C Dubernet, F Nemati, et al. Reversion of multidrug resistance with polyalkylcyanoacrylate nanoparticles:towards a mechanism of action, Br. J. Cancer.1997;76:198-205
[40]A.E. Gulyaev, S.E. Gelperina, I.N. Skidan,et al. Significant transport of doxorubicin into the brain with polysorbate 80-coated nanoparticles, Pharm. Res. 1999;16:1564-9
[41]CE. Soma, C Dubernet, D Bentolila, et al. Reversion of multidrug resistance by co-encapsulation of doxorubicin and cyclosporin A in polyalkylcyanoacrylate nanoparticles, Biomaterials.2000;21:1-7
[42]A Friese, E Seiller, G. Quack, et al. Increase of the duration of the anticonvulsive activity of a novel NMDA receptor an tagonist using poly(butylcyanoacrylate) nanoparticles as a parenteral controlled release system, Eur. J. Pharm. Biopharm. 2000;49:103-9
[43]Kataoka K, Harada A, Nagasaki Y. Block copolymer micelles for drug delivery: design, characterization and biological significance. Adv Drug Deliv Rev.2001; 47:113-31
[44]Torchilin VP, Levchenko TS, Whiteman KR, et al. Amphiphilic poly-N-vinylpyrrolidones:synthesis, properties and liposome surface modification. Biomaterials.2001; 22:3035-44
[45]Otsuka H, Nagasaki Y, Kataoka K et al. PEGylated nanoparticles for biological and pharmaceutical applications. Adv.Drug Deliv Rev.2003;55:403-19
[46]Leamon CP, Low PS. Folate-mediated targeting:from diagnositics to drug and gene delivery. Drug Discov Today,2001,6:44-51
[47]肖苏尧,童春义,刘选明,等.肿瘤靶向性药物载体叶酸-淀粉纳米颗粒的研制与应用.科学通报,2006,51(10)：1151-5
[48]Campbell IG, Jones TA, Foulkes WD, et al. Folate-binding protein is a marker for ovarian cancer, Cancer Res.1991,51:5329-38
[49]Lu Y, Low PS. Immunotherapy of folate receptor-expressing tumors:review of recent advances and future prospects (Review). J Control Release, 2003,91(1):17-29
[50]Bottero F, Tomassetti A, Canevari S, et al. Gene transfection and expression of the ovarian carcinoma marker folate binding protein on NIH/3T3 cells increases cell growth in vitro and in vivo. Cancer Res,1993,53:5791-6
[51]Holm J, Hansen SI, Hoier-Madsen M, et al. Folate receptor of human mammary adenocarcitime, noma. APMIS,1994,102:413-9
[52]Ross JF, Chaudhuri PK, Ratnam M, Differential regulation of folate receptor isoforms in normal and malignant tissuses in vivo and in established cell lines: physiologic and clinical implications. Cancer,1994,73:2431-43
[53]Gruner BA, Weitman SD. The folate receptor as a potential therapeutic anticancer target, Invest. New Drugs,1998,16:205-19
[54]陆伟跃,刘敏,潘俊,等.叶酸-脂质体制备及对Hela细胞靶向作用.上海医科大学学报,2000,27：4-8
[55]Gottschal S, Cristiano RJ, Smith LC, et al. Folate receptor mediated DNA delivery into tumor cells:potosomal disruption results in enhanced gene expression. Gene Ther,1994,1:185-91
[56]Reddy JA, Dean D, Kennedy MD, et al. Optimization of folate-conjugated liposomal vectors for folate receptor-mediated gene therapy. J Pharm Sci, 1999,88(11):1112-8
[57]Teruaki H, Tomohisa F, Shuichi H, et al. Schizophyllan-folate conjugate as a new non-cytotoxic and cancer-targeted antisense carrier. Bioorg Med Chem Lett, 2005,15:327-30
[1]任非,陈建海,陈志良.丝裂霉素C-聚氰基丙烯酸正丁酯磁性纳米球的制备方法及性质研究.中国药学杂志,2005,40(15)：1163-6
[2]任非,陈建海,杨西晓,等.丝裂霉素C聚氰基丙烯酸正丁酯磁性纳米球对肝细胞L-02的生长抑制作用.中国药理学通报,2004,20(10)：1126-30
[3]蔡林,胡治平,牛光峰,等.磁性阿霉素纳米药物研制及其靶向性的实验研究.中国骨肿瘤骨病,2008,7(4)：225-30
[4]Couvreur P, Kante B, Roland M, et al. Polycyanoacrylate nanocapsules as potential lysosomotropic carrier:Preparation, morphological and sorptive properties. J Pham Phamacol,1979,31:331-3
[5]张胜,胡蕴玉,吴道澄.骨形态发生蛋白纳米型缓释制剂的制备及性质.第四军医大学学报,2002,23(18)：1686-9
[6]张志荣,廖工铁.聚氰基丙烯酸酯昊微粒的研究进展.中国药学杂志,1994,29：323-6
[7]Vanthier C, Dubernet C, Fattal E, et al. Poly(alkycyanoacrylates) as biodegradable materials for biomedical applications. Advanced Drug Delivery Review,2003,55:519-48
[8]Schroeder U, Sabel BA, Schroeder H. Diffusion enhancement of drugs by loaded nanoparticles in vitro. Prog Neuropsychopharmacol Biol Psychiatry, 1999,23(5):941-9
[9]马淑燕,欧阳五庆,,刘玉梅,等.以聚氰基丙烯酸正丁酯为载体长春碱纳米粒的制备和性能评价及其对大鼠C6脑神经胶质瘤细胞生长的影响.中国组织工程研究与临床康复,2008,12(19)：3757-60
[10]桂凯,张阳德.载阿霉素聚氰基丙烯酸正丁酯纳米粒对L-02细胞的毒性研究.中国普通外科杂志,2008,17(2)：159-61
[11]Lenaerts V, Couvreur P, Christiaens-Lyyh D, et al. Degradation of poly (isobutyl cyanoncylate) nanoparticles. Biomaterials,1984,5(2):65-8
[12]Muller RH, Lherm C, Herbort J, et al. In vitro modal for the degradation of alkylcyanoacrylate nanoparticles. Biomaterials,1990,11(8):590-5
[13]Kreuter J, Wilson CG, Fry JR, et al. Toxicity and association of polycyanoacrylate nanoparticles with hepatocytes. J Microencapsul, 1984,1(3):253-7
[14]金武龙,周梁,郭慧玲,等.纳米微粒靶向治疗兔舌癌的实验研究.北京口腔医学,2008,16(4)：181-4
[15]Olivier JC. Drug transport to brain with targeted nanoparticles. NeuroRx, 2003,2(1),108-19
[16]SI Park, JH Lim, YH Hwang, et al. In vivo and in vitro antitumor activity of doxorubicin-loaded magnetic fluids. Phy Sstat Sol,2007,12(4):4345-51
[17]姜子荣,杨西晓,兰青.DOX-PBCA-NP的制备及其对脑胶质瘤SHG44细胞的抑制作用观察.山东医药,2008,48(12)：22-4
[18]Phelps ME.PET:The merging of biolog and imaging into molecular imaging. Nucl Med.2000; 41:661-81
[19]Artemov D. Molecula magnetic resonance imaging with targeted contrast agents. J Cell Bio chem.2003; 909:518-24
[20]Heath JR, Phelps ME, HoodL, etal. Nanosystems biology. Mol Imaging Biol. 2003;5:312-25
[21]何勤,张志荣.万乃洛韦聚氰基丙烯酸正丁酯毫微粒制备工艺的优选.华西药学杂志,1998,13(1)：14-6
[22]吕国士,许乙凯.新型MR特异性对比剂Gd-聚氰基丙烯酸正丁酯纳米微粒的合成及其肝脏靶向性作用.中国组织工程研究与临床康复,2008,12(1)：43-6
[23]Giacomo F, Mariano L, Silvana M. Amoxicillin-loaded polyethylcyanoacrylate nanoparticles:Influence of PEG coating on the particle size, drug release rate and phagocytic uptake. Biomaterials,2001,22:2857-65
[24]Gontana G, Potarresi G, Tomarchio V, et al. Preparation characterization and in vitro antimicrobial activity of ampicillin-loaded polyethylcyanoacrylate nanoparticles. Biomaterials,1998,19(11-12):1009
[25]Gregoriadis G. Liposome Technology (Vol 3). Boca Raton:CRC Press, 1984:75-94
[26]张兆旺.重要药剂学.北京：中国中医药出版社,2003：508
[27]Yu Zhang, Siyu Zhu, Lichen Yin, et al. Preparation, characterization and biocompatibility of poly (ethylene glyol)-poly(n-butyl cyanoacrylate) nanocapsules with oil core via miniemulsion polymerization. European Polymer Journal,2008,44:1654-61
[1]Weitman SD, Lark RH, Coney LR, et al. Distribution of the folate receptor GP38 in normal and malignant cell lines and tissuses. Cancer Res,1992,52:3396-401
[2]Lee RJ, Huang L. Folate-targeted, anionic liposome-enliposome-trapped polylysine-condensed DNA for tumor cell-specific gene transfer. J Biol Chem, 1996,271:8481-7
[3]Anderson GB. Folate-binding proteins. Ann Rev Nutr,1990,10:319-35
[4]谢俊玲,沈丹华,杜金荣.叶酸受体α在女性生殖系统肿瘤中的研究进展.中国妇产科临床杂志,2008.9(4)：312-5
[5]Kelemen LE. The role of folate receptor a in cancer development, progression and treatment cause, consequence or innocent bystander. Int J Cancer, 2006,119:243-50
[6]陈慧,李娟.受体介导的肿瘤靶向治疗研究进展.药学与临床研究, 2008,16(4)：288-92
[7]Antony AC. The biological chemistry of folate receptors. Blood, 1992,79(11):2807-20
[8]张寿春,李慧敏,王柯敏.叶酸-聚合物靶向载药系统研究进展.中国医药工业杂志,2007,38(9)：676-80
[9]邢诗安.叶酸受体及其在肿瘤核医学中的应用.国外医学放射医学核医学分册,2002,26(6)：257-9
[10]Konda SD, Aref M, Wang S, et al. Specific targeting of folate-dendrimer MRI contrast agents to the high affinity folate receptor expressed in ovarian tumor xenografts. MAGMA,2001,12(2):104-13
[11]Hoon Choi, Seok, Rye Choi, et al. Iron oxide nanoparticles as magnetic resonance contrast agent for tumor imaging via folate receptor-targeted delivery. Acad Radio.2004,11(9):996-1004
[12]王劲武,黄其鎏,许乙凯等.钆标记抗人大肠癌单克隆抗体CL-3对裸鼠肿瘤模型MRI诊断的实验研究.中华放射学杂志,1998,32(6)：419-22
[13]肖苏尧,童春义,刘选明,等.肿瘤靶向性药物载体叶酸-淀粉纳米颗粒的研制与应用.科学通报,2006,51(10)：1151-5
[14]Campbell IG, Jones TA, Foulkes WD, et al. Folate-binding protein is a marker for ovarian cancer, Cancer Res.1991,51:5329-38
[15]丁建辉,曾蒙苏,薛琼,等.叶酸受体型MR对比剂在荷人胃癌裸鼠诊断价值的实验研究.中华放射学杂志,2005,39(8)：882-6
[1]陈慧,李娟.受体介导的肿瘤靶向治疗研究进展.药学与临床研究,2008,16(4)：288-92
[2]张寿春,李慧敏,王柯敏.叶酸-聚合物靶向载药系统研究进展.中国医药工业杂志,2007,38(9)：676-80
[3]Ross JF, Chaudhuri PK, Ratnam M, Differential regulation of folate receptor isoforms in normal and malignant tissuses in vivo and in established cell lines: physiologic and clinical implications. Cancer,1994,73:2431-43
[4]Weitman SD, Lark RH, Coney LR, et al. Distribution of the folate receptor GP38 in normal and malignant cell lines and tissuses. Cancer Res,1992,52:3396-401
[5]高晓宁,唐锁勤.叶酸受体及其在介导药物靶向肿瘤细胞治疗中的应用.中国实验血液学杂志,2005,13(5)：911-4
[6]Weitman SD, Weinberg AG, Coney LR, et al. Cellular localization of the folate receptor:potential role in drug toxicity and folate homeostasis. Cancer Res, 1992,52:6708-11
[7]Bagnoli M, Canevari S, Figini M, et al. A step further in understanding the biology of the folate receptor in ovarian carcinomar. Gynecol Oncol, 2003,88:S140-4
[8]林凤云,朱照静.叶酸受体介导的靶向给药研究进展.中国医院药学杂志,2006,26(10)：1281-4
[9]Garin-Chesa P, Campbell I, Saigo PE, et al. Trophoblast and ovarian cancer antigen LK26. Sensitivity and specificity in immunapathology and molecular identification as a folate-binding protein. Am J Pathol,1993,142(2):557-67
[10]Cancer Facts and Figures 2007, American Cancer Society, Inc, Atlanta, Georgia, USA (www.cancer.org)
[11]Bueno R, Appasani K, Mercer H, et al. The alpha folate receptor is highly activated in malignant pleural mesothelioma. Thorac. Cardiovasc. Surg, 2001,121(2):225-33
[12]丁建辉,曾蒙苏,薛琼,等.叶酸受体型MR对比剂在荷人胃癌裸鼠诊断价值的实验研究.中华放射学杂志,2005,39(8)：882-6
[13]Bottero F, Tomassetti A, Canevari S, et al. Gene transfection and expression of the ovarian carcinoma marker folate binding protein on NIH/3T3 cells increases cell growth in vitro and in vivo. Cancer Res,1993,53:5791-6
[14]Holm J, Hansen SI, Hoier-Madsen M, et al. Folate receptor of human mammary adenocarcitime, noma. APMIS,1994,102:413-9
[15]Gruner BA, Weitman SD. The folate receptor as a potential therapeutic anticancer target, Invest. New Drugs,1998,16:205-19
[16]陆伟跃,刘敏,潘俊,等.叶酸-脂质体制备及对Hela细胞靶向作用.上海医科大学学报,2000,27：4-8
[17]Gottschal S, Cristiano RJ, Smith LC, et al. Folate receptor mediated DNA delivery into tumor cells:potosomal disruption results in enhanced gene expression. Gene Ther,1994,1:185-91
[18]Reddy JA, Dean D, Kennedy MD, et al. Optimization of folate-conjugated liposomal vectors for folate receptor-mediated gene therapy. J Pharm Sci, 1999,88(11):1112-8
[19]Teruaki H, Tomohisa F, Shuichi H, et al. Schizophyllan-folate conjugate as a new non-cytotoxic and cancer-targeted antisense carrier. Bioorg Med Chem Lett, 2005,15:327-30
[20]Leamon CP, Reddy JA. Folate-targeted chemotherapy. Adv Drug Deliv Rev, 2004,56(8):1127-41
[21]Konda SD, Aref M, Brechbiel M, et al. Development of a tumor-targeting MR contrast agent using the high-affinity folate receptor:work in progress. Invest Radiol,2000,35:50-7
[22]洪国斌,梁碧玲,沈君,等.三维梯度回波磁敏感成像在颅内出血诊断中的初步应用.实用放射学杂志,2008,24(2)：151-4
[23]Natha DW, Judy C, Barbara H, et al. Reliability in detection of hemorrhage in acute stroke by a new three-dimensional gradient recalled echo susceptibility-weighted imaging technique compared to computed tomography: A retrospective study, J Magn Reson Imagin,2994,20(2):372-7
[24]E Mark H, Xu YB, Cheng YC, et al. Susceptibility Weighted Imaging (SWI). Magn Reson Med,2004,52(4):612-8
[25]Wolff SD, Balaban RS. Magnetization transfer imaging:practical aspects and clinical applications. Radiology,1994,192:593-9
[26]Koh DM, Collins DJ. Diffusion-weighted MRI in the body:applications and challenges in oncology. AJR,2007,188(6):1622-35
[27]Gallbraith SM. MR in oncology drug development. NMR Biomed, 2006;19(6):681-9
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |