摘要
目的
1.以葡聚糖-超顺磁氧化铁纳米粒(dextran-superparamagnetic iron oxide nanoparticles, dextran-SPIO-NPs)为阳性对照,对本课题组已经制备好的羧甲基壳聚糖-超小超顺磁氧化铁纳米粒(o-carboxymethyl chitosans ultrasmall superparamagnetic iron oxide nanoparticles, OCMCS-USPIO-NPs)和叶酸-羧甲基壳聚糖-超小超顺磁氧化铁纳米粒(Folic acid-o-carboxymethyl chitosans ultrasmall superparamagnetic iron oxide nanoparticles, FA-OCMCS-USPIO-NPs)进行小鼠急性毒性方面的研究。
2.以dextran-SPIO-NPs为阳性对照,采用紫外-可见分光光度法考察FA-OCMCS-USPIO-NPs和OCMCS-USPIO-NPs在大鼠体内的药物代动力学特征。
3.以dextran-SPIO-NPs为阳性对照,考察FA-OCMCS-USPIO-NPs和OCMCS-USPIO-NPs在小鼠体内的组织分布学特征时设计了两组试验：紫外-可见分光光度法测定组织内的铁含量和核磁共振成像法在体考察药物分布。
4.采用核磁共振成像技术分别评价了FA-OCMCS-USPIO-NPs对BACB/C-nu裸鼠KB细胞移植瘤和OCMCS-USPIO-NPs对新西兰兔VX2淋巴结转移瘤的造影效果。
方法
1.急性毒性的评价
简单介绍了阳性对照dextran-SPIO-NPs和试验药OCMCS-USPIO-NPs与FA-OCMCS-USPIO-NPs的合成方法,主要对两种试验药的急性毒性进行考察。dextran-SPIO-NPs的合成采用碱性共沉淀法：在葡聚糖溶液中合成SPIO-NPs的同时对纳米粒进行包被。分两步合成OCMCS-USPIO-NPs:先采用碱性共沉淀法合成SPIO-NPs核心,再在SPIO-NPs表面嫁接OCMCS。在合成好的OCMCS-USPIO-NPs表面再嫁接以叶酸就合成出了FA-OCMCS-USPIO-NPs。马尔文激光粒径测定仪测定三种纳米粒的水合粒径。急性毒性的考察选择KM小鼠为实验对象,分别尾静脉一次性给予小鼠三个浓度(278,347.5和434.5mgFe·kg-1)的三种纳米粒,观察14d内小鼠的死亡、饮食和体重变化情况。
2.药代动力学评价
SD大鼠禁食12h后(自由饮水),尾静脉一次给予1mL生理盐水及5.87和13.27 mgFe·kg-1的三种纳米粒,并于给药后的0.25,0.5,1,2,4,6,8,12,24h从眼底静脉丛采血0.5mL,至于肝素钠化的EP管中,5000r·mmin-1离心10min,取0.2mL上清液测定铁含量。由于三种纳米粒的活性成分是铁,故采用邻二氮菲法测定血浆内的铁含量。用移液器精密量取血浆样品0.2mL于西林瓶中,加入1mL硝酸-高氯酸(3：1,v：v),室温下消化24h后用平板加热器蒸干,待冷却后加入3%的盐酸溶液1mL溶解西林瓶中的Fe3+离子,并转入10mL容量瓶,分别依次加入10%的盐酸羟胺溶液1mL、0.15%的邻二氮菲溶液2mL、1 mol·L-1 NaAc溶液5 mL,蒸馏水标定,于最大吸收波长处测定吸光度,带入标准曲线方程求出铁含量,再按照稀释比例求算血浆内的铁含量。
3.组织分布学考察
3.1组织铁含量测定
采用邻二氮菲法测定组织内的铁含量。用分析天平称取50mg各组织于西林瓶中,加入1mL混酸(硝酸-高氯酸体积比3：1),室温下消化24h后用平板加热器蒸干,待冷却后加入3%的盐酸溶液1mL溶解西林瓶中的Fe3+离子,并转入10mL容量瓶,分别依次加入10%的盐酸羟胺溶液1mL、0.15%的邻二氮菲溶液2mL、1 mol·L-1 NaAc溶液5 mL,蒸馏水标定,以紫外-可见分光光度法于最大吸收波长处测定吸光度,带入标准曲线方程求出铁含量,再按照稀释比例求算血浆内的铁含量。
KM小鼠禁食12h后(自由饮水),空白对照组尾静脉一次给予0.2mL生理盐水,给药组分别从尾静脉一次给予9.53mg·kg"1或19.06mg·kg-1的三种纳米粒后,分别于2,4,8,16h处死动物(每个时间点3只),取心、肝、脾、肺、肾。用生理盐水洗净组织上的残血,滤纸吸干水分后准确称取50mg,测定各时间点的组织铁含量。
取空白组和16h时高浓度三给药组小鼠的心、肝、脾、肺、肾,于10%福尔马林溶液中固定24h,石蜡包埋并切片,普鲁士蓝染色后,于光学显微镜观察并拍照。
3.2磁共振考察在体分布
SD大鼠禁食12h后(自由饮水),用10%的水合氯醛腹腔注射麻醉。先平扫所有动物,之后所有动物尾静脉一次给予28μg·kg-1的dextran-SPIO-NPs, OCMCS-USPIO-NPs或FA-OCMCS-USPIO-NPs,分别在给药后的1,2,4,6,8,24h进行磁共振扫描。采用头部线圈行T2冠状面扫描,扫描序列参数如下：采用自旋回波-T2加权像(SE-T2WI)扫描序列,重复时间(TR)和回波时间(TE)分别是4000ms和106ms,视野(field of view, FOV):12×12cm,层厚2mm。使用Image Viewer软件测定肝、肺、肾的T2信号值(SI),在背景区域选取较大的区域测定背景噪声的标准差(Standard-deviation of the noise, SD),求出各组织各时间点的信噪比(signal to noise ratio, SNR),计算公式为：SNR=SI/SD,比较给药前和给药后各时间点三种脏器SNR的变化情况。
4.药效学评价
荷KB瘤裸鼠采用头部线圈行T2冠状面扫描,扫描序列参数如下：采用自旋回波-T2加权像(SE-T2WI)扫描序列,重复时间(TR)和回波时间(TE)分别是4000ms和85ms,视野(field of view, FOV):12 X 12cm,层厚3mm。腹腔注射4%的水合氯醛溶液麻醉后平扫所有动物,尾静脉给予5.62 mg·ml-1的FA-OCMCS-SPIO-NPs溶液0.25ml,3h后进行MRI扫描。
VX2淋巴结转移新西兰兔采用头部线圈行T2冠状面扫描,扫描序列参数如下：采用快速白旋回波-T2加权像(FSE-T2WI)扫描序列,重复时间(TR)和回波时间(TE)分别是3500ms和85ms,视野(field of view, FOV):10×10cm,层厚3mm。采用3%的戊巴比妥钠(4℃保存)溶液耳缘静脉注射麻醉后平扫所有动物,耳缘静脉给予2.00mg·ml1的OCMCS-SPIO-NPs溶液16.80ml,给药12h后再进行MRI扫描。
裸鼠肿瘤和新西兰兔淋巴结转移的造影效果分别用信噪比(signal to noise ratio, SNR)和对比噪声比(contrast to noi se ratio, CNR)进行评价。采用MRI阅片软件CDViewer对两组实验动物给药前、后感兴趣区(Region of interest, ROI)的信号强度(signal intense, SI)进行测量,ROI选择局部信号相对均匀,无明显伪影的区域测量三次,取SI平均值。裸鼠给药前、后肿瘤的SNR计算见3.2方法项下,兔淋巴结转移给药前、后CNR计算公式为：CNR=|SInormal-SIcancer| /SDbackground、SInormal,SIcancer和SDbackground分别是淋巴结正常组织信号,淋巴结转移组织信号和背景噪声的标准差。
分别做裸鼠KB肿瘤和兔VX2肿瘤和淋巴结转移瘤的HE染色切片,判断成瘤和转移情况；做给药前、后KB肿瘤和兔VX2肿瘤和淋巴结转移瘤的普鲁士蓝染色切片,判断FA-OCMCS-SPIO-NPs对KB肿瘤的靶向性和OCMCS-SPIO-NPs对兔VX2淋巴结转移瘤的造影效果。
结果
1. dextran-SPIO-NPs, OCMCS-USPIO-NPs和FA-OCMCS-USPIO-NPs的水合粒径分别为125nm,38.2nm和41.4nm。急性毒性结果表明FA-OCMCS-USPIO-NPs和OCMCS-USPIO-NPs的LDso均大于434.5 mgFe·kg-1,小于阳性对照药dextran-SPIO-NPs的LDso(大于347.5 mgFe·kg-1)。两试验药物组中所有动物均未出现明显的毒性反应；dextran-SPIO-NPs组中从中等剂量组开始生存动物出现明显毒性反应。
2.由于药代动力学实验中,基础血浆铁浓度随时间的变化而波动(1h时出现突释峰,之后缓慢下降,4h以后在5mg·L-1左右波动,24h时恢复初始水平),所以,血浆中三种纳米粒的浓度分别以给药后血浆铁浓度减去空白血浆铁浓度表示。结果表明,粒径更小的两试验药在体内半衰期(t1/2)更长(大于7小时),药时曲线下面积更大,体内滞留时间(MRT)显著性延长。
3.五种脏器的铁含量测定结果显示,三种造影剂主要分布于肝、脾部位,但较之于dextran-SPIO-NPs,不论给予低浓度还是高浓度的药物,肝、脾对FA-OCMCS-USPIO-NPs和OCMCS-USPIO-NPs的吞噬量明显减少,并且FA-OCMCS-USPIO-NPs的吞噬也明显少于OCMCS-USPIO-NPs,这可能是因为嫁接叶酸以后纳米粒具有了靶向性,更多的集中于靶组织中,亦或是参与了叶酸途径的代谢,降低了肝、脾途径代谢的量。以上结论可以从三种药物各脏器普鲁士蓝染色切片图上清楚的看到。
磁共振考察肝、肺和肾的结果表明,三种脏器的基础T2信号值不同,由大到小依次为肾>肝>肺。给药后dextran-SPIO-NPs组肝脏信号下降明显,说明肝脏吞噬了纳米粒,使SNR值降低；同时肾脏SNR值降低明显,说明粒径更大的dextran-SPIO-NPs更容易被代谢出体外。肺部以及两试验组肝脏各时间点SNR均值几乎没有变化。24h时dextran-SPIO-NPs组和]FA-OCMCS-USPIO-NPs组的肾脏SNR值恢复至给药前的水平,但OCMCS-USPIO-NPs组只恢复至给药后2-4h的水平,说明OCMCS-USPIO-NPs在体内保留了更久的时间,24h时还有药物从体内排泄,FA-OCMCS-USPIO-NPs'恢复至给药前水平可能是由于嫁接叶酸以后粒径有所增加,加快了体内代谢的过程。
4.药效学结果显示,尾静脉给予FA-OCMCS-USPIO-NPs后裸鼠KB肿瘤T2信号明显降低(FA-OCMCS-USPIO-NPs对有叶酸受体的KB肿瘤有靶向作用),耳缘静脉给予OCMCS-USPIO-NPs后兔胭窝淋巴结正常部位T2信号值下降(淋巴结内的巨噬细胞能吞噬OCMCS-USPIO-NPs),而癌变部位T2信号和给药前一样,几乎没有变化(巨噬细胞失去功能不能再吞噬纳米粒)。统计结果显示,给药前、后KB瘤的SNR有显著性差异(t=11.596,P=0.007),VX2淋巴结转移瘤的CNR有显著性差异(t=10.586,P=0.009),说明两造影剂效果明显。普鲁士蓝染色切片图说明KB瘤内有FA-OCMCS-USPIO-NPs分布(有蓝色颗粒),VX2胭窝转移淋巴结的正常组织内有OCMCS-USPIO-NPs分布(有蓝色颗粒),癌变组织内无纳米粒(无蓝色颗粒)。
结论
1.急性毒性考察结果表明FA-OCMCS-USPIO-NPs和OCMCS-USPIO-NPs的急性毒性小于dextran-SPIO-NPs,这可能是试验药物的粒径(小于50nm)远小于阳性对照药物(大于100nm)造成的,也可能是因为包被材料OCMCS的生物相容性更优于dextran,导致体内毒性下降。
2.药代动力学结果说明大粒径的阳性对照dextran-SPIO-NPs进入体内后迅速被肝、脾吞噬代谢,体内半衰期和AUC较小,而小粒径的OCMCS-USPIO-NPs和FA-OCMCS-USPIO-NPs部分逃避了肝、脾的吞噬,在血液中维持了较长的时间和较高的浓度,为肿瘤靶向造影提供了依据。
3.给药后肝、脾对dextran-SPIO-NPs的吞噬显著高于FA-OCMCS-USPIO-NPs和OCMCS-USPIO-NPs,说明小粒径的两试验药部分逃避了肝、脾内巨噬细胞的吞噬,为二者能分布到全身其他部位进行造影打下了基础；磁共振在体研究三种纳米粒分布代谢中也证实了肝脏对dextran-SPIO-NPs的吞噬,而同等剂量的两试验药没有吞噬,三种药物在肺中没有分布,均从肾脏排泄出体外。
4.药效学结果表明,FA-OCMCS-USPIO-NPs对具有叶酸受体的裸鼠KB细胞移植瘤具有靶向性造影作用,OCMCS-USPIO-NPs对新西兰兔VX2胭窝淋巴结转移瘤具有判断癌灶大小的作用,为有效判断转移性癌灶提供依据。
Objective
1. To research acute toxicity of o-carboxymethyl chitosans ultrasmall superparamagnetic iron oxide nanoparticles (OCMCS-USPIO-NPs) and Folic acid-o-carboxymethyl chitosans ultrasmall superparamagnetic iron oxide nanoparticles (FA-OCMCS-USPIO-NPs) prepared by our group with dextran-superparamagnetic iron oxide nanoparticles (dextran-SPIO-NPs) as a positive control.
2. To study pharmacokinetics features of FA-OCMCS-USPIO-NPs and OCMCS-USPIO-NPs by ultraviolet spectrophotometry with dextran-SPIO-NPs as a positive control.
3. To study in vivo distribution characteristics of FA-OCMCS-USPIO-NPs and OCMCS-USPIO-NPs with dextran-SPIO-NPs as a positive control by two experiments:one was determineing iron content in organs by ultraviolet spectrophotometry, the other was researching distribution in living rats by magnetic resonance imaging.
4. To evaluate imaging results of FA-OCMCS-USPIO-NPs on KB tumor in BACB/C-nu nude mice and the OCMCS-USPIO-NPs on popliteal lymph node metastasis in New Zealand rabbits, respectively.
Methods
1. Acute toxicity evaluation
This part was brief introduction of synthetic methods of dextran-SPIO-NPs, OCMCS-USPIO-NPs and FA-OCMCS-USPIO-NPs, and studying the acute toxicity of OCMCS-USPIO-NPs and FA-OCMCS-USPIO-NPs mainly. The method of alkaline coprecipitation was used to synthesize dextran-SPIO-NPs, and the synthesis of SPIO-NPs was coated in dextran solution at the same times. Synthesis of OCMCS-USPIO-NPs in two steps:synthesis of SPIO-NPs core first, and then grafting OCMCS on the surface of the core. We can synthesize FA-OCMCS-USPIO-NPs by Grafting folic acid on the OCMCS-USPIO-NPs. Determineing hydrated particle size of three nanoparticles using Malvern-3000HS. KM mice were injected three concentrations (278,347.5 and 434.5 mgFe·kg-1) of three nanoparticles from tail vein respectively and observed death, diet and weight during 14 days.
2. Pharmacokinetic evaluation
SD rats were injected 1mL of saline and 5.87 and 13.27 mgFe·kg-1 of three nanoparticles from tail vein respectively after fasted 12h (free water), then we gatherd 0.5 mL of blood from the retinal venous plexus at 0.25,0.5,1,2,4,6,8,12,24 h following administration. Puted the blood in EP tube containing heparin, centrifuged 10 min under the speed of 5000 r·min-1 and mensurated iron content in 0.2 mL of plasma. As the active composition of three nanoparticles was iron, we determined iron content in plasma by phenanthroline method. We pipetted 0.2mL of plasma sample accurately into a vial, added 1mL of nitric acid-perchloric acid (3:1, v:v), and evaporated by the heater after digestion 24h at room temperature.1 mL of 3% Hydrochloric acid solution were added into the vial to dissolve Fe3+ completely after the vail was cool, then we transferred the solution to volumetric flask (10mL). We added 1mL of 10% hydroxylamine hydrochloride solution,2mL of 0.15% phenanthroline solution and 5mL of 1 mol·L-1 NaAc solution into the volumetric flask, calibrated using distilled water, and measured the absorbance at the maximum absorption wavelength, then putted the absorbance into the standard curve to calculate the iron content of plasma.
3. In vivo distribution evaluation
3.1 determination iron content in tissues
We determined iron content in tissues by phenanthroline method. We weighed 50mg of organization accurately into a vial using analytical balance, added 1mL of nitric acid-perchloric acid (3:1, v:v), and evaporated by the heater after digestion 24h at room temperature.1 mL of 3% Hydrochloric acid solution were added into the vial to dissolve Fe3+ completely after the vail was cool, then we transferred the solution to volumetric flask (10mL). We added 1mL of 10% hydroxylamine hydrochloride solution,2mL of 0.15% phenanthroline solution and 5mL of 1 mol·L-1 NaAc solution into the volumetric flask, calibrated using distilled water, and measured the absorbance at the maximum absorption wavelength, then putted the absorbance into the standard curve to calculate the iron content of plasma.
KM mice were injected 1mL of saline in control group and 9.53mg·kg-1 or 19.06mg·kg-1 of three nanoparticles in treatment group from tail vein respectively after fasted 12h (free water), then we executed animals at 2,4,8,16h (each time point 3) following administration. Heart, liver, spleen, lung and kidney of mice were taked, washed with saline, exsiccated with filter paper, weighed 50mg accurately, and measured iron content.
Heart, liver, spleen, lung and kidney of mice in control and high concentration group at 16h were fixed in 10% formalin solution, embedded with paraffin, cutted into slices, stained with Prussian blue and photographed under optical microscope.
3.2 In vivo distribution study by MRI
SD rats were anesthetized with 10% chloral hydrate solution by intraperitoneal injection after fasted 12h (free water). We Scaned all the animals before administration, then injected 28μg·kg-1 of dextran-SPIO-NPs, OCMCS-USPIO-NPs or FA-OCMCS-USPIO-NPs from tail vein respectively. All the animals were scaned at 1,2,4,6,8,24 h after injection by MRI. We scaned at coronal section with head coil to get the T2 signal value, and scan sequence parameters were as follows:We used spin echo-T2 weighted (SE-T2WI) sequences, repetition time (TR) and echo time (TE) were 4000ms and 106ms, field of vision (Field of view, FOV) was 12×12cm, slice thickness was 2mm. The T2 signal value (SI) of liver, lung and kidney and standard deviation of background noise (SD) were measured using the Image Viewer software, then calculated and compared signal to noise ratio (SNR) of three tissues at different time points. The formula is:SNR=SI/SD.
4. pharmacodynamic evaluation
KB tumor-bearing nude mice were scaned at coronal section with head coil to get the T2 signal value, and scan sequence parameters were as follows:We used spin echo-T2 weighted (SE-T2WI) sequences, repetition time (TR) and echo time (TE) were 4000ms and 85ms, field of vision (Field of view, FOV) was 12×12cm, slice thickness was 3mm. We anesthetized all the animals with 4% chloral hydrate solution by intraperitoneal injection and Scaned them before administration. KB tumor-bearing nude mice were injected 5.62 mg·ml-1 of the FA-OCMCS-SPIO-NPs solution (0.25mL) from tail vein respectively, and scaned following 3h.
New Zealand white rabbits with lymph node metastasis of VX2 tumor were scaned at coronal section with head coil to get the T2 signal value, and scan sequence parameters were as follows:We used fast spin echo-T2 weighted (FSE-T2WI) sequences, repetition time (TR) and echo time (TE) were 4000ms and 85ms, field of vision (Field of view, FOV) was 12×12cm, slice thickness was 3mm. We anesthetized all the animals with 3% pentobarbital sodium (4℃save) solution by ear vein injection and Scaned them before administration. New Zealand white rabbits with lymph node metastasis of VX2 tumor were injected 2.00 mg·ml-1 of the OCMCS-SPIO-NPs solution (0.25mL) from tail vein respectively, and scaned following 12h.
Using SNR and contrast to noise ratio (CNR) evaluated contrast effect of KB tumor-bearing nude mice and New Zealand white rabbits with lymph node metastasis of VX2 tumor separately. The T2 SI of Region of interest (ROI) before and after administration and SD of background noise were measured using the Image Viewer software. We selected ROI with a homogeneous signal and measured SI three times. The average of SI was used finally to calculate and the SNR of KB tumor and CNR of lymph node metastasis of VX2 tumor. Calculation method of SNR see paragraph 3.2. The formula of CNR is CNR=| SInormal-SIcancer |/SDbackground-SInormal, SIcancer and SDbackground were SI of normal lymph node, SI of lymph node metastasis and background noise standard deviation.
We maked the HE staining of KB tumor in nude mice and VX2 tumor and lymph node metastases in New Zealand white rabbits to determine the formation of the tumor and the situation of metastasis. We maked the Prussian blue staining of KB tumor in nude mice and VX2 tumor and lymph node metastases in New Zealand white rabbits to determine the imaging results of the FA-OCMCS-SPIO-NPs on the KB tumor and OCMCS-SPIO-NPs on lymph node metastasis of VX2 tumor.
Results
1. The hydrated particle size of dextran-SPIO-NPs, FA-OCMCS-USPIO-NPs and OCMCS-USPIO-NPs was 125,38.2 and 41.4nm. Acute toxicity results showed that the LD50 of FA-OCMCS-USPIO-NPs and OCMCS-USPIO-NPs was greater than 434.5 mgFe·kg-1 which was less than it of dextran-SPIO-NPs (greater than 347.5 mgFe·kg-1). All the animals in two test groups were not found obvious toxicity, but significant toxicity was appeared in middle and high dose groups of dextran-SPIO-NPs.
2. Because the basical plasma iron concentration fluctuated at the different times in pharmacokinetic study (one peak of plasma iron concentration was appeared at 1h, then the concentration was decreased slowly and fluctuated in the vicinity of 5mg·L-1 from 4h. It resumed the initial level at 24h), plasma drug concentration of three nanoparticles was expressed by the differentials between plasma iron concentration in treatment groups and blank plasma iron concentration. The results showed that two test drugs with smaller size had longer half-life (t1/2 was more than 7h), larger area under the curve and the mean residence time (MRT) extended significantly.
3. Determination of iron content in five organs showed that three contrast agents mainly existed in liver and spleen, but the quantity of FA -OCMCS-USPIO-NPs and OCMCS-USPIO-NPs was swallowed by liver and spleen was less than dextran-SPIO-NPs whether in high or low concentration groups. Moreover, the phagocytosis of FA-OCMCS-USPIO-NPs was smaller than OCMCS-USPIO-NPs, which may be due to the graft of folic acid making FA-OCMCS-USPIO-NPs possessing targeting; it made FA-OCMCS-USPIO-NPs focuse on tissues with folic acid receptor. It is also probably because FA-OCMCS-USPIO-NPs involves in the metabolism of folate pathway, which reduces metabolism from liver and spleen. These conclusions could be seeing in the Prussian blue staining of organs clearly.
The resaults of In vivo distribution of three nanoparticles in liver, lung and kidney by MRI showed that the basical T2 signal value of three organs was different, from high to low was kidney, liver and lung. Liver signal in dextran-SPIO-NPs group decreased significantly after administration, as liver swallowed dextran-SPIO-NPs making SNR values decrease. SNR values of kidneys in dextran-SPIO-NPs group decreased significantly, as larger particle size of dextran-SPIO-NPs was easily metabolized out of the body. Mean SNR values of lung in three groups and liver in two test groups had little change. SNR values of kidneys in dextran-SPIO-NPs and FA-OCMCS-USPIO-NPs groups were returned to the normal level at 24h, but it only returned to the level of 2-4h after administration in OCMCS-USPIO-NPs group, as OCMCS-USPIO-NPs retained in the body longer time and excreted from body at 24h after administration. SNR values of kidneys in FA-OCMCS-USPIO-NPs groups were returned to the normal level may be due to graft of folic acid enlargeing the size and speeding up the body metabolic process.
4. Pharmacodynamic results showed that the T2 signal of KB tumor in nude mice reduced after intravenous injection of FA-OCMCS-USPIO-NPs (FA-OCMCS-USPIO-NPs could target KB tumor with folate receptors). The T2 signal of normal part in the VX2 popliteal lymph node metastasis in New Zealand white rabbits reduced after intravenous injection of OCMCS-USPIO-NPs (macrophages in lymph node could swallow OCMCS-USPIO-NPs), while the cancerous part had no change compared with preinjection (macrophages losed function and could not swallow nanoparticles). Statistical results show that SNR value of KB tumor had significant difference (t=11.596, P=0.007) and CNR value of VX2 popliteal lymph node metastasis had significant difference (t=10.586, P=0.009) illustrating the imaging effect of two contrast agents was obvious. Prussian blue staining showed that there were FA-OCMCS-USPIO-NPs in the KB tumor and OCMCS-USPIO-NPs in normal part in the VX2 popliteal lymph node metastasis (having blue stain), but there was no OCMCS-USPIO-NPs in cancerous part in the VX2 popliteal lymph node metastasis (having on blue stain).
Conclusions
1. The results of acute toxicity study showed that the acute toxicity of FA-OCMCS-USPIO-NPs and OCMCS-USPIO-NPs was lower than dextran-SPIO-NPs, which is probably because the particle size of two test drugs (less than 50nm) was much smaller than the positive drug (greater than 100nm) or the biocompatibility of coated materials (OCMCS) was better than dextran decreasing the toxicity.
2. pharmacokinetic results indicated that positive drug (dextran-SPIO-NPs) with large particle size was quickly swallowed and metabolized by liver and spleen in vivo owning shorter half-life and smaller AUC, while OCMCS-USPIO-NPs and FA-OCMCS-USPIO-NPs with small particle size could partly escape from phagocytosis of liver and spleen maintaining longer time and higher concentration in vivo. It provided a basis for target-imaging cancer.
3. The phagocytosis of liver and spleen of dextran-SPIO-NPs was more significant than FA-OCMCS-USPIO-NPs and OCMCS-USPIO-NPs after administration, which illustrated OCMCS-USPIO-NPs and FA-OCMCS-USPIO-NPs with small particle size could partly escape from phagocytosis of liver and spleen providing a basis for imaging all of the body. The resaults of In vivo distribution of three nanoparticles by MRI confirmed the phagocytosis of dextran-SPIO-NPs in liver and spleen, but no phagocytosis of two test drugs. Three drugs were not found in lung and excreted from the kidney.
4. pharmacodynamic results show that FA-OCMCS-USPIO-NPs could target-imaging KB tumor transplanted in nude mice whose surface had folate receptors and OCMCS-USPIO-NPs could used to judgment of cancer size in the VX2 popliteal lymph node metastasis in New Zealand white rabbits providing a basis for judgment of metastatic focus.
引文
[1]Claire Corot, Philippe Robert, Jean-Marc Idee, et al. Recent advances in iron oxide nanocrystal technology for medical imaging [J].Advanced Drug Delivery Reviews, 2006,58:1471-1504.
[2]Conroy Sun, Jerry S.H. Lee, Miqin Zhang. Magnetic nanoparticle in MR imaging and drug delivery[J].Advanced Drug Delivery Reviews,2008,60:1252-1265.
[3]Bauer W R, Schulten K. Theory of contrast agents in magnetic resonance imaging: coupling of spin relaxation and transport [J]. Magn Reson Med,1992,26(1):16-39.
[4]Sviridov N K, Napolov I, Bolotova E N, et al. Use of ultrasmall ferric oxide (Sinerem) particles as a magnetic resonance contrast substance for imaging lymph nodal metastases in cancer of the head and neck [J]. Vestn Rentgenol Radiol,2004(3): 63-64.
[5]杨汉丰,周翔平。菲立磁增强肝脏磁共振成像[J].川北医学院学报,2004(03).
[6]Robert S. Molday, Vancouver. Magnetic iron-dextran microspheres.1984, US patent 4452773, Section8.
[7]Ernest V. Groman, Lee Josephson. Biologically degradable superparamagnetic particles for use as nuclear magnetic resonance imaging agents.1988, US patent 4770183, Section6.1.
[8]刘世霆,晏媛,陈志良,等。超顺磁性葡聚糖氧化铁纳米颗粒的研制及表征[J]南方医科大学学报,2006；26(3)：331-334.
[9]M.G. Harisinghani, J. Barentsz, P.F. Hahn, et al. Noninvasive detection of clinically occult lymph-node metastases in prostate cancer[J]. N. Engl. J. Med,2003, 348:2491-2499.
[10]J.M. Rogers, J. Lewis, L. Josephson. Visualization of superior mesenteric lymph nodes by the combined oral and intravenous administration of the ultrasmall superparamagnetic iron oxide AMI-227 [J]. Magn. Reson. Imaging,1994,12: 1161-1165.
[11]Y. Anzai, S. McLachlan, M. Morris, et al. Dextran-coated superparamagnetic iron oxide an MR contrast agent for assessing lymph nodes in the head and neck [J]. Am. J. Neuroradiol,1994,15:87-94.
[12]M.F. Bellin, C. Roy, K. Kinkel, et al. Lymph node metastases:safety and effectiveness of MR imaging with ultrasmall superparamagnetic iron oxide particles-initial clinical experience [J]. Radiology,1998,207:799-808.
[13]Mai Russell, Yoshimi Anzai. Ultrasmall superparamagnetic iron oxide enhanced MR imaging for lymph node metastases[J]. Radiography,2007,13:e73-84.
[14]R. Weisssleder, A. Bogdnanov, E.A. Neuwelt, et al. Long circulating iron oxides for MR imaging [J]. Adv. Drug Deliv. Rev,1995,13:321-334.
[15]M.F. Bellin, C Beigelman, S. Precetti-Morel. Iron oxide-enhanced MR lymphography:initial experience[J]. Eur. J. Radiol,2000,34:257-264.
[16]J. W. Bulte, D.L. Kraitchman. Iron oxide MR contrast agents for molecular and cellular imaging [J]. NMR Biomed,2004,17:484-499.
[17]H.W. Kang, L. Josephson, A. Petrovsky, et al. Magnetic resonance imaging of inducible E-selectin expression in human endothelial cell culture [J]. Bioconjug. Chem,2002,13:122-127.
[18]I. Pirko, A. Johnson, B. Ciric [J]. In vivo magnetic resonance imaging of immune cells in the central nervous system with superparamagnetic antibodies [J]. FASEB,2004,18:179-181.
[19]L. Josephson, C.H. Tung, A. Moore, et al. High-efficiency intracellular magnetic labeling with novel superparamagnetic-Tat peptide conjugates [J]. Bioconjug. Chem, 1999,10:186-191.
[20]Sun C, Lee JSH, Zhang MQ. Magnetic nanoparticle in MR imaging and drug delivery [J].Advanced Drug Delivery Reviews,2008,60 (11):1252-1265.
[21]何跃明。恶性肿瘤的磁靶向热疗[J].国外医学.物理医学与康复学分册,2003(02).
[22]Latorre M, Rinaldi C. Applications of magnetic nanoparticles in medicine: magnetic fluid hyperthermia. [J]. P R Health Sci J,2009,28(3):227-238.
[23]Caixia Fan, Wenhui Gao, Zhixi Chen, et al. Tumor selectivity of stealth multi-functionalized superparamagnetic iron oxide nanoparticles[J].international journal of pharmaceutics.2011,404(1-2):180-190.
[1]Caixia Fan, Wenhui Gao, Zhixi Chen, et al. Tumor selectivity of stealth multi-functionalized superparamagnetic iron oxide nanoparticles[J].international journal of pharmaceutics.2011,404(1-2):180-190.
[2]范彩霞,高文慧,陈志良,等。超小的羧甲基壳聚糖超顺磁氧化铁纳米粒制备及处方优化[J].中国现代应用药学,2010,27(9)：825-831.
[3]Molday, RS, MacKenzie, D. Immunospecific ferromagnetic iron-dextran reagents for the labeling and magnetic separation of cells [J]. Immunol.1982,52(3),353-367.
[4]王国斌,夏泽锋,陶凯雄,等。医用纳米级Fe304磁流体的急性毒理学实验研究[J].华中科技大学学报(医学版),2004,33(4)：52-454,458.
[5]Ralph Weissleder, David D. Stark, and Barry L. Engelstad, et al. Superparamagnetic Iron oxide:Pharmacokinetics and Toxicity [J]. AJR.1989,152: 167-173.
[6]胡润磊,柯贤福,李浒,等。磁流体体内毒性的初步实验研究[J].实用医学杂 志,2010,26(12)：2114-2116.
[7]文明,宋琳,柏玮,等。纳米级超顺磁性氧化铁的制备及其对小鼠急性毒性作用的观察[J].第二军医大学学报,2007,28(10)：1104-1108.
[8]中华人民共和国卫生部药政局新药(西药)临床前研究指导原则汇编(药学药理学毒理学)[S].北京：卫生部,1993：199.
[9]Claire Corot, Philippe Robert, Jean-Marc Idee, et al. Recent advances in iron oxide nanocrystal technology for medical imaging [J].Advanced Drug Delivery Reviews,2006,58:1471-1504.
[10]范彩霞,高文慧,陈志良,等。体外评价羧甲基壳聚糖超顺磁氧化铁纳米粒的细胞毒性和巨噬细胞摄取[J].华西药学杂志,2010,25(3)：290-293.
[1]Ma HL, Xu YF, Qi XR, et al. Superparamagnetic iron oxide nanoparticles stabilized by alginate:Pharmacokinetics, tissue distribution, and applications in detecting liver cancers[J]. Int J Pharm,2008,354(1-2):217-266.
[2]孙敬方,主编。动物实验方法学[M].第一版.北京：人民卫生出版社,2001：357.
[3]Plassat V, Martina M.S, Barratt G, et al. Sterically stabilized superparamagnetic liposomes for MR imaging and cancer therapy:Pharmacokinetics and biodistribution[J]. Int J Pharm,2007,344(1-2):118-127.
[4]Kai C, Jin X, Hengyi X, et al. Triblock copolymer coated iron oxide nanoparticle conjugate for tumor integrin targeting[J]. Biomaterials,2009,30:6912-6919.
[5]Peter A.J, Torjus S, Anita G, et al. Iron oxide core oil-in-water emulsions as a multifunctional nanoparticle platform for tumor targeting and imaging[J]. Biomaterials,2009,30:6947-6954.
[6]王彩霞,李春雷,赵曦,等。盐酸米托蒽醌脂质体的药效学、药动学及组织分布[J].药学学报,2010,45(12)：1565-1569.
[7]吴燕,张福成,吴诚,等。盐酸表阿霉素长循环热敏脂质体大鼠药代动力学考察[J].药学学报,2010,45(3)：365-370.
[8]冯亮,胡昌江,余凌英。人参皂苷Rgl及其代谢产物的药代动力学研究[J]. 药学学报,2010,45(5)：636-640.
[9]Corot C, Robert P, Jean-Marc I, et al. Recent advances in iron oxide nanocrystal technology for medical imaging [J].Adv Drug De,2006,58(14):1471-1504.
[10]Plassat V, Martina M.S, Barratt G, et al. Sterically stabilized superparamagnetic liposomes for MR imaging and cancer therapy:Pharmacokinetics and biodistribution [J]. Int J Pharm,2007,344(1-2):118-127.
[11]刘金玉,张玉,王凯平。当归多糖铁大鼠体内药动学参数与给药剂量的关系[J].中国医院药学杂志,2010,30(3)：183-185.
[12]姚小平,涂干卿。Ferene-s络合法检测血清铁的方法学评价[J].现代医药卫生,2007,23(3)：347-348.
[13]岳志劲,关翠林,王海清,等。分光光度法测定家兔体内铁和锰的代谢[J].光谱实验室,2010,27(4)：1607-1609.
[1]Ma HL, Xu YF, Qi XR, et al. Superparamagnetic iron oxide nanoparticles stabilized by alginate:Pharmacokinetics, tissue distribution, and applications in detecting liver cancers[J]. Int J Pharm,2008,354(1-2):217-266.
[2]Akihiro T, Sachio K. Hepatocyte-targeted MR contrast agents:contrast enhanced detection of liver cancer in diffusely damaged liver[J]. Magnetic Resonance in Medical Sciences,2005,4(2):53-60.
[3]Akihiro T, Makio M, Sachio K.Evaluation of superparamagnetic iron oxide for MR Imaging of liver injury:proton relaxation mechanisms and optimal MR imaging parameters[J]. Magn Reson Med Sci,2006,5 (2):89-98.
[4]孙敬方,主编。动物实验方法学[M].第一版.北京：人民卫生出版社,2001：357.
[5]Bernd H, Matthias T, Petra H, et al. MR Lymphography with iron oxide particles: dose-response studies and pulse sequence optimization in rabbits [J]. HAMM ET AL, 1992,158:183-190.
[6]徐戎,师少军,周顺长,等。5-氟尿嘧啶磁性白蛋白亚微球在正常及荷瘤小鼠体内的药代动力学与组织分布[J].药学学报,2007,42(1)：66-70.
[7]Molday RS, MacKenzie D. Immunospecific ferromagnetic iron-dextran reagents for the labeling and magnetic separation of cells [J]. Immunol,1982,52(3),353-367.
[1]Caixia Fan, Wenhui Gao, Zhixi Chen, et al. Tumor selectivity of stealth multi-functionalized superparamagnetic iron oxide nanoparticles[J].international journal of pharmaceutics.2011,404(1-2):180-190.
[2]Garin-Chesa P, Campbell I, Saigo PE, et al. Trophoblast and ovarian cancer antigen LK26. Sensitivity and specificity in immu2 nopat hology and molecular identification as a folate2binding protein [J]. Am J Pat hol,1993,142 (2):557-567.
[3]Bueno R, Appasani K, Mercer H, et al. The alpha folate receptor is highly activated in malignant pleural mesot helioma [J].Thorac.Cardiovasc.Surg,2001,121 (2):225-233.
[4]Weitman SD, Weinberg AG, Coney LR, et al. Cellular localization of the folate receptor:potential role in drug toxicity and folate homeostasis [J]. Cancer Res,1992, 52 (23):6708-6711.
[5]Ross J F, Chaudhufi P K, Ratnam M. Differential regulation of folate receptor isoforms in normal and malignant tissues in vivo and in established cell lines: Physiologic and clinical implications [J]. Cancer,1994,73 (9):2432-2443.
[6]林凤云,朱照静。叶酸受体介导的靶向给药研究进展[J].中国医院药学杂志,2006,26(10)：1281-1284.
[7]Kai C, Jin X, Hengyi X, et al. Triblock copolymer coated iron oxide nanoparticle conjugate for tumor integrin targeting[J]. Biomaterials,2009,30:6912-6919.
[8]Tomoaki H, Nobuhiko T, Mitsuru M, et al. Evaluation of lymph node metastases of breast cancer using ultrasmall superparamagnetic iron oxide-enhanced magnetic resonance imaging [J]. European Journal of Radiology,2007,63:401-407.
[9]Corot C, Robert P, Jean-Marc I, et al. Recent advances in iron oxide nanocrystal technology for medical imaging [J].Adv Drug De,2006,58(14):1471-1504.
