【作者】 李欢；

【导师】 徐海波；

【作者基本信息】 华中科技大学 ， 影像医学与核医学， 2015， 博士

【摘要】 目的：制备FA-BSA修饰的高分子聚合物磁性胶束作为肿瘤特异性靶向磁共振对比剂,用于肿瘤靶向磁共振成像。方法：1.合成：(1)采用化学共沉淀法及油酸修饰制备单分散的磁性纳米粒子(SPIONs);(2)采用自由基聚合法合成阳离子两亲高分子聚合物(poly(HFMA-co-MOTAC)-g-PEGMA);(3)采用自组装技术制备阳离子高分子聚合物磁性胶束(简称“未修饰的磁性胶束”)：(4)阳离子磁性胶束与FA-BSA通过静电相互作用最终合成FA-BSA修饰的磁性胶束。2.表征：(1)使用’H NMR观察两亲高分子聚合物的化学组成,使用FTIR观察磁性纳米粒子、高分子聚合物和FA-BSA修饰的磁性胶束的化学结构；(2)电镜观察磁性胶束的物理形态和大小,DLS检测磁性胶束的水合粒径及分布；(3)使用热重分析磁性胶束的载铁含量,绘制磁滞回线观察所制备胶束的磁性特点；(4)用3T磁共振扫描机及腕关节线圈,采用多回波T2WI磁共振成像方法检测磁性胶束的横向弛豫率。3.体外细胞实验：(1)使用MTT法检测磁性胶束的细胞毒性；(2)细胞摄取实验：将不同浓度的FA-BSA修饰的磁性胶束和未修饰的磁性胶束分别与人肝癌细胞Bel-7402(叶酸受体阳性表达)和Hep3B(叶酸受体阴性表达)共培养,其中未修饰的磁性胶束作为对照,分别采用磁共振成像、细胞铁含量测定、普鲁士蓝染色的方法,对于两种肿瘤细胞对两种磁性胶束的摄取量进行定性、定量及病理分析。4.体内实验：(1)制备裸鼠肝癌皮下瘤模型；(2)载瘤动物活体磁共振成像：尾静脉注射四种不同的造影剂,包括T1对比剂Gd-DTPA,T2对比剂Feridex,以及合成的未修饰磁性胶束与FA-BSA修饰的磁性胶束,观察所制备的FA-BSA修饰的磁性胶束对载瘤动物活体磁共振成像的效果。其中钆、菲立磁与单纯磁性胶束作为对照。(3)组织化学分析：对肝脏和肿瘤组织进行铁特异性普鲁士蓝染色,定性分析磁性胶束在肝脏和肿瘤组织中的分布和含量；(4)肿瘤组织铁定量测定,载瘤裸鼠尾静脉注射未修饰磁性胶束与FA-BSA修饰的磁性胶束,24h后定量测定肿瘤组织的铁含量。结果：1.成功合成未修饰的磁性胶束与FA-BSA修饰的磁性胶束；2.表征：(1)1H NMR, FTIR结果从化学结构上证明磁性胶束合成成功；(2)电镜显示所合成的磁性胶束呈球形的壳-核结构,DLS测得所合成的未修饰磁性胶束与FA-BSA修饰磁性胶束平均水合粒径分别为161.5nm(PDI=0.21)、196.1nm(PDI=0.26);(3)热重分析未修饰磁性胶束与FA-BSA修饰磁性胶束的氧化铁含量分别为27.5wt%、19.2wt%；磁滞回线测得二者的饱和磁化强度分别为13.9emu/g、5.5emu/g;(4)采用多回波T2WI磁共振扫描分析测定未修饰的磁性胶束与FA-BSA修饰的磁性胶束的横向弛豫率分别为243.9mM-1s-1、179.5mM-1s-1。3.体外细胞实验：(1)MTT法检测不同浓度两种磁性胶束(从25μg/mL到100μg/mL)与正常肝细胞孵育24,48h,细胞活性均在80%以上,说明两种磁性胶束没有细胞毒性,可应用于生物医学；(2)细胞摄取实验,无论是叶酸受体阳性的Bcl-7402细胞,还是叶酸受体阴性的Hep3B细胞,两者对FA-BSA修饰的磁性胶束的摄取均高于未修饰的磁性胶束；但是对于FA-BSA修饰的磁性胶束,Bel-7402的摄取量高于Hep3B细胞对其的摄取量：而对未修饰的磁性胶束,两种肿瘤细胞Bel-7402、Hep3B的摄取率基本相同：Hep3B、Bel-7402细胞对未修饰的磁性胶束摄取率分别为(6.174±0.19)%,(6.43±0.45)%：Hep3B、Bel-7402细胞对FA-BSA修饰的磁性胶束摄取率分别为(8.83±0.41)%,(12.07+1.29)%。4.体内实验：(1)尾静脉注射对比剂后1h-24h, FA-BSA修饰的磁性胶束实验组皮下瘤T2信号降低最明显,而对照组(未修饰的磁性胶束及Feridex)肿瘤信号改变程度较小。(2)肝脏普鲁士蓝染色结果表明Feridex在肝脏沉积较多,未修饰的磁性胶束在肝脏沉积较少,而FA-BSA修饰的磁性胶束在肝脏无明显沉积。肿瘤普鲁士蓝染色结果表明FA-BSA修饰的磁性胶束在肿瘤组织的聚集明显高于未修饰的磁性胶束：而Feridex组仅仅在肿瘤组织血管内可以看到少许蓝色颗粒,作为阴性对照Gd-DTPA组在肿瘤组织没有看到蓝色颗粒。(3)肿瘤组织铁含量测定：肿瘤组织对未修饰的磁性胶束和FA-BSA修饰的磁性胶束的平均摄取率分别为(1.54±0.041)%,(2.91±0.035)%。结论：1.成功制备高分子磁性胶束与FA-BSA修饰的磁性胶束；2.体外和体内实验均说明FA-BSA修饰的磁性胶束具有很强的磁共振成像能力,并且能显著增加肿瘤细胞与肿瘤组织对磁性胶束的摄取,可以作为高表达叶酸受体的肿瘤的靶向对比剂；3.未修饰的高分子磁性胶束也有一定的肿瘤靶向作用和磁共振成像效果。更多还原

【Abstract】 Purpsoe:FA-BSA modified polymeric magnetic micelles are investigated as a specific contrast agent for tumor targeting and magnetic resonance imaging in vitro and in vivo.Methods:1. reparation:(1) Mono-dispersed SPIONs were synthesized by chemical co-precipitation and modified with oleic acid.(2) The cationic amphiphilic poly(HFMA-co-MOTAC)-g-PEGMA copolymers were synthesized by free radical polymerization.(3) Cationic polymeric magnetic micelles denoted as unmodified magnetic micelles were prepared by self-assembly.(4) FA-BSA modified magnetic micelles were synthesized by functionalizing the cationic magnetic micelles with FA-BSA by electrostatic complexation.2. Characterization:(1)1H NMR was conducted to investigate the chemical structure of the amphiphilic poly(HFMA-co-MOTAC)-g-PEGMA copolymers. The structures of the SPIONs, amphiphilic poly(HFMA-co-MOTAC)-g-PEGMA copolymers, and FA-BSA modified magnetic micelles were assessed by FTIR.(2) The morphology of the magnetic micelles was examined by TEM at200kV. The hydrodynamic size and size distribution were measured using DLS.(3) The thermogravimetric analysis was performed on the Perkin Elmer TGA-7. The magnetic properties were studied on a VSM at298K under an applied magnetic field.(4) The transverse relaxivity (r2) of the magnetic micelles was determined using a3.0-T whole body MR scanner in combination with an8-channel wrist joint coil and tested by T2-weighted multi-echo spin echo sequence.3. In-vitro studies:(1) The cytotoxicity of the magnetic micelles was estimated by means of the MTT assay.(2) Cell uptaken studies:The human hepatoma cell lines Bel-7402(FR-positive) and Hep3B (FR-negative) were incubated with the FA-BSA modified magnetic micelles or unmodified magnetic micelles dissolved in DMEM at different iron concentrations. The unmodified magnetic micelles were used as controls. The uptake of both the FA-BSA modified and unmodified magnetic micelles is evaluated by different methods including MR imaging, intracellular iron content determination, and iron-specific Prussian blue staining. The uptaken results were carried out quantitative, qualitative and pathological analysises, respectively.4. In-vivo studies:(1) Establishment of human hepatoma xenografts in nude mice.(2) In vivo MRI:Different contrast agents including the T1contrast agent Gd-DTPA, T2contrast agents Feridex, unmodified magnetic micelles, and FA-BSA modified magnetic micelles were used via tail vein administration, in order to investigate the feasibility of the FA-BSA modified magnetic micelles in specific MR imaging of tumors. The first three were used as control groups.(3) Histochemistry analysis:the magnetic micelles in the liver and tumor tissues of the aforementioned tumor-bearing nude mice were evaluated by Iron-specific Prussian blue staining.(4) Tumor iron content determination:The uptake percentages of the unmodified and FA-BSA modified magnetic micelles in tumor tissue were determined by Spectr AA240FS at24h after tail vein administration.Results:1. The unmodified magnetic micelles and FA-BSA modified magnetic micelles were prepared successfully.2. Characterization:(1) The results of’H NMR and FTIR confirmed the chemical structure and construction of unmodified magnetic micelles and FA-BSA modified magnetic micelles.(2) Both of unmodified and FA-BSA modified magnetic micelles were spherical core-shell structures tested by TEM. The average hydrodynamic particle size of unmodified and FA-BSA modified magnetic micelles tested by DLS were about161.5nm (PDI=0.26),196.1nm (PDI=0.26), respectively.(3) The content in the unmodified and FA-BSA modified magnetic micelles were calculated to be27.5wt%and19.2wt%, respectively. The saturation magnetization values of the unmodified and FA-BSA modified magnetic micelles were13.9emu/g and5.5emu/g, respectively.(4) The transverse relaxivity (r2) of the unmodified and FA-BSA modified magnetic micelles tested by multi-echo T2-weighted MR imaging were243.9mM-1s-1and179.5mM-1s-1, respectively.3. In-vitro studies:(1) Cell viability of over80%was maintained from the human normal liver cell line HL-7702after incubation for24h and48h by increasing the iron concentration from25μg/mL to100μg/mL, indicating that both the FA-BSA modified and unmodified magnetic micelles have no obvious cytotoxicity and are suitable probes in biomedical applications.(2) Cell uptaken results:Both FR-positive Bel-7402cells and FR-negative Hep3B cells showed enhanced cellular uptake of FA-BSA modified magnetic micelles comparing with unmodified magnetic micelles. But the average uptake rate of FA-BSA modified magnetic micelles in Bel-7402cells was higher than Hep3B cells. The uptake of unmodified magnetic micelles in Bel-7402was similar to that in Hep3B cells. The average uptake percentages of unmodified magnetic micelles by Hep3B cells and Bel-7402cells were calculated to be (6.17±0.19)%,(6.43±0.45)%, respectively. The average uptake percentages of FA-BSA modified magnetic micelles by Hep3B cells and Bel-7402cells were (8.83±0.41)%,(12.07±1.29)%, respectively.4. In-vivo studies:(1) The T2signal intensity of tumor in the group treated with the FA-BSA modified magnetic micelles decreases most significantly between1h to24h. While the tumors with Feridex and unmodified magnetic micelles show minimal contrast change in the same period.(2) Prussian blue staining performed on the group with Feridex shows large accumulation of magnetic particles in the liver, whereas the group with unmodified magnetic micelles shows less blue spots. In contrast, the FA-BSA modified magnetic micelles do not accumulate much in the liver. Prussian blue staining of the tumor tissues was performed at24h post-injection of contrast agents. Accumulation of blue spots in the tumor tissues observed from the folate-targeted groups is obviously larger than that of untargeted groups. There are a small number of blue spots trapped in the tumor vascularity in the groups of Feridex and no visible blue spots in the groups of Gd-DTPA.(3) Tumor iron content determination: The uptake percentages of the unmodified and FA-BSA modified magnetic micelles in tumor tissue were calculated to be (1.54±0.041)%,(2.91±0.035)%, respectively.Conclusions:1. Polymer magnetic micelles and FA-BSA modified magnetic micelles are successfully synthetized.2. Both In vitro and in vivo studies demonstrate that FA-BSA modified magnetic micelles have excellent MR imaging effect and high selectivity and sensitivity to FR-positve hepatoma cells and tissue, indicating their potential use as a negative targeted MRI contrast agent for folate-receptor overexpressing cancer.3. The unmodified magnetic cells also maintain good tumor targeting and MR imaging ability.更多还原