本文選題：焦糖納米球　切入點：氧化錳　出處：《山東大學(xué)》2017年碩士論文

【摘要】：研究背景原發(fā)性肝癌,是起源于肝細(xì)胞或肝內(nèi)膽管上皮細(xì)胞的惡性腫瘤,屬于我國常見的惡性腫瘤之一。流行病學(xué)研究顯示,肝癌在全球癌癥發(fā)病率中位列第3,我國肝癌患病率位居世界第1,居我國癌癥死因的第2位。目前肝癌的最佳治療方式是手術(shù)切除,因此早期影像診斷對于肝癌尤為重要。肝癌的影像診斷方法主要包括磁共振成像(MRI)、計算機(jī)斷層掃描(CT)、正電子發(fā)射斷層掃描(PET)及超聲檢查(US)。而MRI具有無創(chuàng)檢查、三維成像和高密度分辨率的優(yōu)勢,在肝癌診斷中備受關(guān)注。對比劑可增加MRI圖像對比度,從而提高影像診斷率。對比劑可分為順磁性對比劑和超順磁性對比劑兩種,以釓和錳為代表的順磁性對比劑主要用于T1WI成像,臨床以馬根維顯最為常用;以鐵為代表的超順磁性對比劑主要用于T2WI成像,以SPIO最為常見。多次應(yīng)用釓基對比劑可導(dǎo)致腎纖維化,同時SPIO應(yīng)用于T2WI使圖像信號減低可能丟失圖像信息,因此錳基對比劑引起了國內(nèi)外學(xué)者的關(guān)注。雖然錳具有神經(jīng)系統(tǒng)和循環(huán)系統(tǒng)毒性,但是經(jīng)研究發(fā)現(xiàn)低劑量氧化錳不足以導(dǎo)致帕金森病和心臟毒性。另外,錳離子的核外不成對電子較多,T1加權(quán)效果強(qiáng)于釓離子。右旋糖苷是由葡萄糖組成的親水多聚糖,具有良好的生物兼容性,作為大分子物質(zhì)可以延長藥物的循環(huán)時間,主要用于藥物和蛋白質(zhì)的轉(zhuǎn)運。此外,右旋糖酐的代謝主要在細(xì)胞溶酶體中降解,少量經(jīng)葡糖聚酶降解和小膽管排泄。研究表明,右旋糖酐分子量越大,其代謝越快�；诖�,本研究制備了氧化錳-焦糖納米球用于肝膽惡性腫瘤的MRI T1加權(quán)成像。其具備以下特征:1、焦糖納米球作為運載體,可延長氧化錳的循環(huán)時間,從而實現(xiàn)長效延遲強(qiáng)化;2、焦糖納米球可通過細(xì)胞內(nèi)溶酶體降解,具備潛在的pH響應(yīng)性,實現(xiàn)對比劑可降解腫瘤靶向特性;3、腫瘤EPR效應(yīng)、長效循環(huán)和pH響應(yīng)可躲避肝脾內(nèi)網(wǎng)狀內(nèi)皮細(xì)胞的吞噬,實現(xiàn)肝膽惡性腫瘤MR T1加權(quán)成像。研究目的驗證長效可降解的氧化錳-焦糖納米球(Mn@CNS)應(yīng)用于肝膽惡性腫瘤磁共振T1加權(quán)成像的可行性并探討其腫瘤增強(qiáng)機(jī)制,以提高肝膽惡性腫瘤的特異性診斷。研究方法1.納米球構(gòu)建與表征:以葡萄糖單體為原料,通過水熱合成法制備焦糖化納米球,再通過氧化還原反應(yīng)與高錳酸鉀偶聯(lián),制備氧化錳-焦糖納米球。用透射電鏡(TEM)觀察納米球大小,用紅外光譜分析納米球表面功能基團(tuán);通過MR T1 Mapping和T2Tapping測試納米球的弛豫率r1和r2,并且與Gd-DTPA(馬根維顯)的弛豫率比較。2.體外實驗:通過細(xì)胞毒性實驗CCK8測試Mn@CNS對人肝癌細(xì)胞系HepG2的活力影響;通過電感耦合等離子體質(zhì)譜(ICP-MS)測定小鼠巨噬細(xì)胞系RAW264.7、大鼠肝細(xì)胞系BRL-3A與人肝癌細(xì)胞系HepG2對Mn@CNS的攝取量;通過流式細(xì)胞儀測試右旋糖酐硫酸酯鈉是否對異硫氰酸熒光素標(biāo)記(FITC)的Mn@CNS存在競爭性抑制;通過Western Blot測定RAW264.7、BRL-3A、HepG2攝入Mn@CNS過程中清道夫受體(ScR)表達(dá)量的變化;通過TEM觀察RAW264.7、BRL-3A、HepG2對Mn@CNS攝取途徑;通過激光共聚焦顯微鏡觀察Mn@CNS在RAW264.7、BRL-3A、HepG2三種細(xì)胞內(nèi)細(xì)胞器定位。3.動物實驗:通過亞硝基二乙胺(DEN)與硫代乙酰胺(TAA)分別誘導(dǎo)SD大鼠原位肝癌與肝內(nèi)膽管癌,肝癌組經(jīng)尾靜脈注射Mn@CNS多時間點觀察腫瘤強(qiáng)化效果,肝內(nèi)膽管癌組進(jìn)一步證實Mn@CNS強(qiáng)化效果的可重復(fù)性,肝內(nèi)膽管癌組經(jīng)尾靜脈注射優(yōu)維顯(Gd-EOB-DTPA)與Mn@CNS對比延遲強(qiáng)化特點;處死大鼠,解剖肝臟,TEM觀察不同時間點大鼠肝膽惡性腫瘤對Mn@CNS排泄途徑;Western Blot測定Mn@CNS對Balb/c小鼠肝內(nèi)錳代謝相關(guān)蛋白二價金屬離子受體(DMT-1)和轉(zhuǎn)鐵蛋白受體(TfR)的影響;PAS染色觀察大鼠肝癌對焦糖化右旋糖酐代謝變化。結(jié)果1.TEM結(jié)果顯示CNS和Mn@CNS的直徑分別為140nm和160nm,并且納米粒表面有黑色物質(zhì)被覆,表明氧化錳與焦糖化右旋糖酐已成功耦合。紅外光譜顯示Mn@CNS的峰值是3300cm-1,表明Mn@CNS表面含有羥基。T1 Mapping與T2 Mapping結(jié)果顯示Mn@CNS的r1及r2分別為11.63和41.36 mM-1s-1,而馬根維顯的r1及r2分別為4.11和4.82 mM-1s-1。,表明Mn@CNS比馬根維顯有較高的弛豫率。2.細(xì)胞毒性CCK8實驗顯示HepG2與不同濃度(25,50,100,200 μg/mL)的Mn@CNS共培養(yǎng)12 h,24h,36 h,48h,細(xì)胞活力介于80%至120%之間,表明Mn@CNS沒有明顯細(xì)胞毒性。3.細(xì)胞攝取實驗ICP-AES測得RAW264.7,BRL-3A和HepG2在攝入Mn@CNS前后的錳離子量分別為 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/細(xì)胞,表明錳離子攝取量HepG2RAW264.7BRL-3A。4.競爭性抑制實驗結(jié)果顯示從1倍至50倍增加右旋糖酐硫酸酯鈉的量,RAW264.7對FITC-Mn@CNS攝入量明顯降低,表明右旋糖酐硫酸酯鈉對ScR存在競爭性抑制。5.ScR的Western Blot結(jié)果顯示Mn@CNS可明顯上調(diào)RAW264.7內(nèi)ScR的表達(dá)(p0.05),而BRL-3A和HepG2無明顯上調(diào)作用,表明RAW264.7依賴ScR攝入納米粒,而BRL-3A和HepG2可能通過其他方式攝入納米粒。6.細(xì)胞TEM顯示RAW264.7,BRL-3A和HepG2可在不同時間點經(jīng)胞吞攝入Mn@CNS,形成囊泡(核內(nèi)體),最終與溶酶體融合降解,表明三種細(xì)胞均可攝取納米粒,最終經(jīng)溶酶體降解。7.激光共聚焦顯微鏡顯示FITC-Mn@CNS在RAW264.7,BRL-3A和HepG2內(nèi)最終定位于溶酶體。8.大鼠MRI結(jié)果表明大鼠原位肝癌及肝內(nèi)膽管癌可明顯延遲強(qiáng)化達(dá)1d,強(qiáng)化高峰出現(xiàn)在注藥后4h;注射優(yōu)維顯后大鼠肝內(nèi)膽管癌僅延遲強(qiáng)化2h,肝膽期出現(xiàn)于45min。9.PAS染色顯示注藥后3h內(nèi)肝癌內(nèi)Mn@CNS較高,而1d后基本恢復(fù)至初始狀態(tài),表明Mn@CNS可經(jīng)過溶酶體降解。10.肝癌和肝內(nèi)膽管癌TEM顯示Mn@CNS被攝入肝癌細(xì)胞內(nèi)并且小膽管內(nèi)未見Mn@CNS,表明Mn@CNS主要經(jīng)溶酶體分解排泄。11.TfR及DMT-1的Western Blot結(jié)果顯示Balb/c小鼠肝內(nèi)DMT-1明顯上調(diào)(p=0.002),而TfR未見明顯上調(diào),表明肝內(nèi)錳的代謝轉(zhuǎn)運主要依靠DMT-1。結(jié)論通過水熱合成法成功構(gòu)建了氧化錳-焦糖納米球,具有T1弛豫率高、細(xì)胞毒性小、可降解及長效延遲的特點。納米粒主要被肝癌細(xì)胞及巨噬細(xì)胞攝取,在溶酶體內(nèi)降解排泄,并且納米球可躲避巨噬細(xì)胞的攝取,積聚于腫瘤細(xì)胞。T1加權(quán)MRI顯示腫瘤經(jīng)氧化錳-焦糖納米球注射后明顯強(qiáng)化,延遲強(qiáng)化時間明顯高于肝膽特異對比劑優(yōu)維顯。
[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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735;R445.2

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4 王帥;張寧;葛小鵬;萬瑛博;李曉虹;嚴(yán)禮;夏怡君;宋波;;多組分反應(yīng)制備規(guī)整偶氮苯聚合物:光致形變性能的超分子納米球[A];中國化學(xué)會第29屆學(xué)術(shù)年會摘要集——第08分會：高分子科學(xué)[C];2014年

5 程軻;王書杰;程剛;蔣曉紅;杜祖亮;;利用納米球模板技術(shù)組裝周期性排列的空心金球[A];中國化學(xué)會第26屆學(xué)術(shù)年會超分子組裝與軟物質(zhì)材料分會場論文集[C];2008年

6 耿，

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