长效可降解氧化锰—焦糖纳米球用于肝胆恶性肿瘤MR增强及机制研究

发布时间：2018-04-04 14:42

本文选题：焦糖纳米球　切入点：氧化锰　出处：《山东大学》2017年硕士论文

【摘要】：研究背景原发性肝癌,是起源于肝细胞或肝内胆管上皮细胞的恶性肿瘤,属于我国常见的恶性肿瘤之一。流行病学研究显示,肝癌在全球癌症发病率中位列第3,我国肝癌患病率位居世界第1,居我国癌症死因的第2位。目前肝癌的最佳治疗方式是手术切除,因此早期影像诊断对于肝癌尤为重要。肝癌的影像诊断方法主要包括磁共振成像(MRI)、计算机断层扫描(CT)、正电子发射断层扫描(PET)及超声检查(US)。而MRI具有无创检查、三维成像和高密度分辨率的优势,在肝癌诊断中备受关注。对比剂可增加MRI图像对比度,从而提高影像诊断率。对比剂可分为顺磁性对比剂和超顺磁性对比剂两种,以钆和锰为代表的顺磁性对比剂主要用于T1WI成像,临床以马根维显最为常用;以铁为代表的超顺磁性对比剂主要用于T2WI成像,以SPIO最为常见。多次应用钆基对比剂可导致肾纤维化,同时SPIO应用于T2WI使图像信号减低可能丢失图像信息,因此锰基对比剂引起了国内外学者的关注。虽然锰具有神经系统和循环系统毒性,但是经研究发现低剂量氧化锰不足以导致帕金森病和心脏毒性。另外,锰离子的核外不成对电子较多,T1加权效果强于钆离子。右旋糖苷是由葡萄糖组成的亲水多聚糖,具有良好的生物兼容性,作为大分子物质可以延长药物的循环时间,主要用于药物和蛋白质的转运。此外,右旋糖酐的代谢主要在细胞溶酶体中降解,少量经葡糖聚酶降解和小胆管排泄。研究表明,右旋糖酐分子量越大,其代谢越快。基于此,本研究制备了氧化锰-焦糖纳米球用于肝胆恶性肿瘤的MRI T1加权成像。其具备以下特征:1、焦糖纳米球作为运载体,可延长氧化锰的循环时间,从而实现长效延迟强化;2、焦糖纳米球可通过细胞内溶酶体降解,具备潜在的pH响应性,实现对比剂可降解肿瘤靶向特性;3、肿瘤EPR效应、长效循环和pH响应可躲避肝脾内网状内皮细胞的吞噬,实现肝胆恶性肿瘤MR T1加权成像。研究目的验证长效可降解的氧化锰-焦糖纳米球(Mn@CNS)应用于肝胆恶性肿瘤磁共振T1加权成像的可行性并探讨其肿瘤增强机制,以提高肝胆恶性肿瘤的特异性诊断。研究方法1.纳米球构建与表征:以葡萄糖单体为原料,通过水热合成法制备焦糖化纳米球,再通过氧化还原反应与高锰酸钾偶联,制备氧化锰-焦糖纳米球。用透射电镜(TEM)观察纳米球大小,用红外光谱分析纳米球表面功能基团;通过MR T1 Mapping和T2Tapping测试纳米球的弛豫率r1和r2,并且与Gd-DTPA(马根维显)的弛豫率比较。2.体外实验:通过细胞毒性实验CCK8测试Mn@CNS对人肝癌细胞系HepG2的活力影响;通过电感耦合等离子体质谱(ICP-MS)测定小鼠巨噬细胞系RAW264.7、大鼠肝细胞系BRL-3A与人肝癌细胞系HepG2对Mn@CNS的摄取量;通过流式细胞仪测试右旋糖酐硫酸酯钠是否对异硫氰酸荧光素标记(FITC)的Mn@CNS存在竞争性抑制;通过Western Blot测定RAW264.7、BRL-3A、HepG2摄入Mn@CNS过程中清道夫受体(ScR)表达量的变化;通过TEM观察RAW264.7、BRL-3A、HepG2对Mn@CNS摄取途径;通过激光共聚焦显微镜观察Mn@CNS在RAW264.7、BRL-3A、HepG2三种细胞内细胞器定位。3.动物实验:通过亚硝基二乙胺(DEN)与硫代乙酰胺(TAA)分别诱导SD大鼠原位肝癌与肝内胆管癌,肝癌组经尾静脉注射Mn@CNS多时间点观察肿瘤强化效果,肝内胆管癌组进一步证实Mn@CNS强化效果的可重复性,肝内胆管癌组经尾静脉注射优维显(Gd-EOB-DTPA)与Mn@CNS对比延迟强化特点;处死大鼠,解剖肝脏,TEM观察不同时间点大鼠肝胆恶性肿瘤对Mn@CNS排泄途径;Western Blot测定Mn@CNS对Balb/c小鼠肝内锰代谢相关蛋白二价金属离子受体(DMT-1)和转铁蛋白受体(TfR)的影响;PAS染色观察大鼠肝癌对焦糖化右旋糖酐代谢变化。结果1.TEM结果显示CNS和Mn@CNS的直径分别为140nm和160nm,并且纳米粒表面有黑色物质被覆,表明氧化锰与焦糖化右旋糖酐已成功耦合。红外光谱显示Mn@CNS的峰值是3300cm-1,表明Mn@CNS表面含有羟基。T1 Mapping与T2 Mapping结果显示Mn@CNS的r1及r2分别为11.63和41.36 mM-1s-1,而马根维显的r1及r2分别为4.11和4.82 mM-1s-1。,表明Mn@CNS比马根维显有较高的弛豫率。2.细胞毒性CCK8实验显示HepG2与不同浓度(25,50,100,200 μg/mL)的Mn@CNS共培养12 h,24h,36 h,48h,细胞活力介于80%至120%之间,表明Mn@CNS没有明显细胞毒性。3.细胞摄取实验ICP-AES测得RAW264.7,BRL-3A和HepG2在摄入Mn@CNS前后的锰离子量分别为 18.69± 1.27,20.03±1.13,11.71 ±1.22 和 21.51 ±0.8,38.17 ± 1.93,63.67 ± 3.54 pg/细胞,表明锰离子摄取量HepG2RAW264.7BRL-3A。4.竞争性抑制实验结果显示从1倍至50倍增加右旋糖酐硫酸酯钠的量,RAW264.7对FITC-Mn@CNS摄入量明显降低,表明右旋糖酐硫酸酯钠对ScR存在竞争性抑制。5.ScR的Western Blot结果显示Mn@CNS可明显上调RAW264.7内ScR的表达(p0.05),而BRL-3A和HepG2无明显上调作用,表明RAW264.7依赖ScR摄入纳米粒,而BRL-3A和HepG2可能通过其他方式摄入纳米粒。6.细胞TEM显示RAW264.7,BRL-3A和HepG2可在不同时间点经胞吞摄入Mn@CNS,形成囊泡(核内体),最终与溶酶体融合降解,表明三种细胞均可摄取纳米粒,最终经溶酶体降解。7.激光共聚焦显微镜显示FITC-Mn@CNS在RAW264.7,BRL-3A和HepG2内最终定位于溶酶体。8.大鼠MRI结果表明大鼠原位肝癌及肝内胆管癌可明显延迟强化达1d,强化高峰出现在注药后4h;注射优维显后大鼠肝内胆管癌仅延迟强化2h,肝胆期出现于45min。9.PAS染色显示注药后3h内肝癌内Mn@CNS较高,而1d后基本恢复至初始状态,表明Mn@CNS可经过溶酶体降解。10.肝癌和肝内胆管癌TEM显示Mn@CNS被摄入肝癌细胞内并且小胆管内未见Mn@CNS,表明Mn@CNS主要经溶酶体分解排泄。11.TfR及DMT-1的Western Blot结果显示Balb/c小鼠肝内DMT-1明显上调(p=0.002),而TfR未见明显上调,表明肝内锰的代谢转运主要依靠DMT-1。结论通过水热合成法成功构建了氧化锰-焦糖纳米球,具有T1弛豫率高、细胞毒性小、可降解及长效延迟的特点。纳米粒主要被肝癌细胞及巨噬细胞摄取,在溶酶体内降解排泄,并且纳米球可躲避巨噬细胞的摄取,积聚于肿瘤细胞。T1加权MRI显示肿瘤经氧化锰-焦糖纳米球注射后明显强化,延迟强化时间明显高于肝胆特异对比剂优维显。
[Abstract]:The research background is the origin of primary carcinoma of the liver, liver cells or intrahepatic bile duct epithelial cells of malignant tumors, is one of the most common malignant tumors in China. Epidemiological studies have shown that liver cancer incidence in the world ranked third in China, the prevalence of liver cancer ranked first in the world, cancer-related death in China at present second. The best way of treatment of liver cancer is surgical resection, so early diagnosis for liver cancer is particularly important. Imaging method in diagnosis of HCC include magnetic resonance imaging (MRI), computer tomography (CT), positron emission tomography (PET) and ultrasound (US). MRI is a noninvasive, three-dimensional imaging and high the density resolution has attracted much attention in the diagnosis of hepatocellular carcinoma. Contrast agent MRI can increase the image contrast, so as to improve the diagnostic rate of contrast agent can be divided into paramagnetic contrast agent and superparamagnetic of agent two, and with gadolinium Mn as the representative of the paramagnetic contrast agent is mainly used for T1WI imaging, clinical Magnevist is most commonly used in iron; as the representative of the super paramagnetic contrast agent is mainly used for T2WI imaging, SPIO is the most common. Many applications of gadolinium based contrast agent can lead to renal fibrosis, and the application of SPIO in T2WI may reduce the image signal the loss of image information, so the manganese based contrast agent attracted the attention of scholars at home and abroad. Although manganese has the nerve system and circulatory system toxicity, but the study found that low dose of manganese oxide is not enough to cause Parkinson's disease and cardiac toxicity. In addition, the manganese ions outside the nuclear unpaired electrons more, better than T1 weighted gadolinium ion. Dextran hydrophilic polysaccharide is composed of glucose, with good biocompatibility, as macromolecules can prolong the circulation time of drugs, mainly for drug and protein trafficking. In addition, dextrose The main degradation in cell metabolism anhydride in lysosomes, a small amount of glucose by enzymatic degradation and the biliary excretion of polymer. The results show that the higher the molecular weight of dextran, its metabolism faster. Based on this, this study prepared manganese oxide nanoparticles for MRI - Caramel T1 weighted imaging of malignant hepatobiliary tumor. It has the following characteristics 1, caramel nanoparticles as a carrier, can prolong the cycle time of manganese oxide, so as to realize the long delayed enhancement; 2, caramel nanospheres by intracellular lysosomal degradation, have the potential to achieve pH response, contrast agent can degrade tumor targeting of tumor; 3, EPR effect, long-term cycle and pH avoid the reticuloendothelial phagocytic response in the liver and spleen, the malignant hepatobiliary tumor T1 weighted imaging. MR objectivestudy manganese oxide long-acting biodegradable Caramel nanospheres (Mn@CNS) applied to the feasibility of hepatobiliary neoplasm magnetic resonance imaging with T1 right And to explore the mechanism of tumor enhancement, in order to improve the specificity of diagnosis of malignant hepatobiliary tumor. Methods 1.: Construction and characterization of nanoparticles with glucose monomers as raw materials, preparation of caramel nanoparticles by hydrothermal synthesis method, and then through the redox coupling reaction with Potassium Permanganate, the preparation of manganese oxide nanoparticles by transmission - Caramel. Electron microscopy (TEM) observation of nanoparticles size, functional analysis of nanoparticles surface groups by infrared spectroscopy; R1 and R2 by MR T1 and T2Tapping Mapping rate test nanospheres and relaxation, and Gd-DTPA (Ma Genwei) the relaxation rate is.2. in vitro: the effects of CCK8 cell cytotoxicity test on human hepatocellular carcinoma cell Mn@CNS HepG2 Department of energy; by inductively coupled plasma mass spectrometry (ICP-MS) determination of mouse macrophage cell line RAW264.7, the amount of uptake of Mn@CNS and BRL-3A human hepatocellular carcinoma cell line HepG2 in rat liver cells by flow cytometry; The test of whether dextran sulfate sodium fluorescein isothiocyanate (FITC) labeled Mn@CNS competitive inhibition; Determination of RAW264.7 by Western Blot BRL-3A, HepG2 Mn@CNS in the process of intake of scavenger receptor (ScR) expression changes observed by TEM; RAW264.7, BRL-3A, HepG2 on Mn@CNS uptake pathway; by laser confocal microscopy. Mn@CNS in RAW264.7, BRL-3A, HepG2 three kinds of organelle positioning.3. through animal experiment: two nitroso ethylamine (DEN) amide and TAA (TAA) were induced in SD rat orthotopic liver cancer and liver bile duct cancer, liver cancer group by intravenous injection of Mn@CNS at different time points to observe the tumor enhancement effect, intrahepatic cholangiocarcinoma further confirmed the effect of Mn@CNS enhanced repeatability of intrahepatic cholangiocarcinoma group by tail vein injection Ultravist (Gd-EOB-DTPA) compared with Mn@CNS delayed enhancement characteristics; the rats were sacrificed and the anatomy of the liver, TEM observation At the same time excretion pathway in Mn@CNS rats liver malignant tumor; Western Blot determination of Mn@CNS related protein on manganese metabolism in the liver of mice Balb/c two valent metal ion receptor (DMT-1) and transferrin receptor (TfR) effect; observation of rat liver of dextran on the metabolic changes of caramel PAS staining. Results 1.TEM results showed that CNS and Mn@CNS the diameter is respectively 140nm and 160nm, and the surface of the nanoparticles with black material coating, showed that the manganese oxide and caramelized dextran has been successfully coupled. The infrared spectra showed that the peak of Mn@CNS is 3300cm-1, Mn@CNS showed that the surface hydroxyl containing Mapping and.T1 T2 Mapping showed that R1 and R2 Mn@CNS were 11.63 and 41.36 mM-1s-1, R1 and R2 and the horse Genwei was respectively 4.11 and 4.82 mM-1s-1., showed that Mn@CNS had higher than Magnevist relaxation rate of.2. cell toxicity experiments showed that HepG2 and CCK8 of different concentration (25,50100200. G/mL) Mn@CNS were cultured for 12 h, 36 24h, h, 48h, cell viability ranged from 80% to 120%, Mn@CNS showed no obvious cytotoxic.3. cell uptake experiments of ICP-AES measured by RAW264.7, BRL-3A and HepG2 in the intake of manganese ion before and after Mn@CNS were 18.69 + 1.27,20.03 + 1.13,11.71 + 1.22 and 21.51 + 0.8,38.17. 1.93,63.67 + 3.54 pg/ cells showed that the manganese ion uptake of HepG2RAW264.7BRL-3A.4. competitive inhibition experiment showed that the increase of dextran sulfate sodium from 1 times to 50 times, RAW264.7 intake of FITC-Mn@CNS decreased significantly, indicating that the right rotation of dextran sulfate sodium on ScR competitive inhibition of.5.ScR Western Blot showed that Mn@CNS expression was up-regulated RAW264.7 ScR (P0.05), while BRL-3A and HepG2 had no obvious effect on ScR showed that RAW264.7 up-regulated, intake of nanoparticles, while BRL-3A and HepG2 may be through the way he intake TEM nanoparticles.6. cells showed RAW264.7, BRL-3A and HepG2 at different time points after intake of Mn@CNS endocytosis, vesicle formation (the nucleus), the final fusion with lysosomes degradation, showed that three kinds of cells can uptake of the nanoparticles, and ultimately by the lysosomal degradation of.7. laser confocal microscope showed that FITC-Mn@CNS in RAW264.7, BRL-3A and HepG2 in the final position in lysosomes of rat.8. MRI results showed that the in situ rat liver in hepatocellular carcinoma and cholangiocarcinoma was significantly delayed enhancement of 1D, strengthen the peak at 4h after injection; injection Ultravist rats after intrahepatic cholangiocarcinoma only delayed enhancement 2h, hepatobiliary phase appears in 45min.9.PAS staining showed higher Mn@CNS 3H injection in hepatocellular carcinoma after the treatment, and after 1D returned to the initial state, indicated that Mn@CNS after lysosomal degradation of.10. liver and intrahepatic bile duct cancer TEM display Mn@CNS ingested hepatoma cells and small bile duct showed no Mn@CNS, Mn@CNS The decomposition of.11.TfR and DMT-1 excretion of lysosomal Western Blot results showed that Balb/c DMT-1 was up-regulated in the liver of mice (p=0.002), and TfR was significantly up-regulated in the liver metabolism showed that manganese mainly depends on DMT-1. conclusion through hydrothermal method successfully constructed manganese oxide nanoparticles with caramel, T1 relaxation rate, cell low toxicity, biodegradable and long delay characteristics. Nanoparticles mainly by hepatoma cells and macrophages in the lysosomal degradation and excretion of nanoparticles can avoid macrophage uptake and accumulation at the tumor cell.T1 weighted MRI showed tumor by manganese oxide - Caramel nanospheres after injection enhancement, delayed enhancement time was significantly higher than that of liver specific contrast agent Ultravist.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735;R445.2

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