基于负载USPIO脂质纳米粒肝组织双靶向分子影像实验研究
发布时间：2018-08-02 21:44
【摘要】：固体脂质纳米粒(Solid lipid nanoparticles, SLN)是近年来正在发展的一种新型纳米粒给药系统,以固态的天然或合成的类脂如单硬脂酸甘油酯、卵磷脂、三酰甘油等为载体,将药物或造影剂包裹于类脂核中制成酸态给药体。因其表面的强疏水性,静脉注射进入机体后,很快被网状内皮系统(Reticuloendothelial system, RES)吞噬而被动进入肝脾,具有显著的被动肝靶向作用。去唾液酸糖蛋白受体(Asialoglycoprotein receptor, ASGPR)是一种完全由哺乳动物肝窦状隙的肝实质细胞特异性表达的膜表面蛋白,能够特异性地识别带有半乳糖残基的糖蛋白,每个肝细胞表面有多达5105个受体,而在肝炎、肝硬化、肝癌或肝转移癌等肝脏疾病时,其数量和功能均有所下降。半乳糖是肝细胞表面ASGPR的特异性配体,是肝靶向基团,具有诱导和提高肝细胞在细胞外基质支架材料上的粘附性能。本研究选择以SLN作为载体,利用SLN自身的肝脏被动靶向特征,结合肝脏的主动靶向修饰技术,制备了经半乳糖修饰的以SLN为包被材料的对肝具有双靶向功能的脂质磁性纳米粒Gal-SLN-USPIO,并在体外和体内实验中考察了其对正常肝细胞、肝癌细胞及正常肝组织的靶向作用,以及对原位移植肝癌模型的诊断价值。具体研究内容主要包括以下四个部分： 第一部分：脂质磁性纳米粒的制备及理化性质研究 本研究首先采用乳糖酸(LA)和硬脂胺(ODA)之间的化学耦合反应制备半乳糖-硬脂胺嫁接物(Gal-ODA),采用核磁共振氢谱(1H NMR)和傅里叶变换红外光谱(FTIR)对其化学结构进行确证；在此基础上,以单甘脂为脂质材料,采用水性溶剂扩散法分别制备对肝脏具有被动靶向功能的脂质磁性纳米粒(SLN-USPIO)、主动靶向功能的脂质磁性纳米粒(Gal-SLN-USPIO/PEG)及双重靶向功能的脂质磁性纳米粒(Gal-SLN-USPIO),其中SLN-USPIO和Gal-SLN-USPIO/PEG作为对照组,并对三种脂质磁性纳米粒的理化性质进行了评价。采用微粒粒度及表面电位分析仪测定三种不同的脂质磁性纳米粒的粒径、表面电位及其分布,采用透射电镜(TEM)观察脂质磁性纳米粒的形态。研究结果表明,本研究所制备得到的三种脂质磁性纳米粒数均粒径在45~60nm之间,表面电位在-27～-35mV之间,多分散指数在0.14～0.35之间,三者之间无显著性差异；脂质磁性纳米粒均呈拟球形,大小均匀,表面平滑完整,颗粒分散性良好,常温放置2周后未见明显聚集现象；外加强磁场吸附实验考察证实脂质纳米粒中均包裹有磁性Fe304纳米颗粒。 第二部分：脂质磁性纳米粒的体外细胞实验及在体正常肝组织靶向性研究 本研究采用异硫氰酸荧光素(FITC)标记脂质磁性纳米粒；以小鼠单核/巨噬细胞RAW264.7为被动靶向细胞模型,人正常肝细胞L02为主动靶向细胞模型和人肝癌细胞HepG2为疾病细胞模型,采用激光共聚焦显微镜定性和流式细胞计数仪定量研究三种细胞对不同脂质磁性纳米粒的摄取情况；采用四甲基偶氮唑盐微量酶反应比色法(MTT实验)考察不同的脂质磁性纳米粒分别对三种细胞的毒性；采用脂溶性DiR作为近红外荧光染料,制备DiR标记磁性脂质纳米粒,采用小动物活体荧光成像仪观察不同的脂质磁性纳米粒在正常裸鼠肝脏的分布。研究结果表明：RAW264.7细胞对SLN-USPIO的摄取强于Gal-SLN-USPIO和Gal-SLN-USPIO/PEG, LO2细胞对Gal-SLN-USPIO的摄取强于SLN-USPIO和Gal-SLN-USPIO/PEG,而HepG2细胞对三种脂质磁性纳米粒的摄取无明显差异性；L02和HepG2两种细胞共孵育竞争性摄取实验表明,L02细胞对Gal-SLN-USPIO的摄取明显强于HepG2细胞对Gal-SLN-USPIO的摄取,这一研究结果为后续的在体肝癌模型分子成像及诊断提供了可能；三种脂质磁性纳米粒在浓度达到100μg/ml时,各种细胞的存活率均在80%以上,说明本研究所制备的脂质磁性纳米粒具有较低的毒性,对临床应用具有可靠的安全性；在体肝脏分布研究结果表明,对肝脏具有双靶向功能的Gal-SLN-USPIO在不同时间点的肝脏分布均强于SLN-USPIO和Gal-SLN-USPIO/PEG对照组,该结果提示具有双靶向功能的Gal-SLN-USPIO有望成为肝脏疾病诊断的磁共振造影剂。 第三部分：脂质磁性纳米粒的体外细胞MR成像研究 配制7管铁浓度分别为0、1、10、15、25、50,100μg/ml的Gal-SLN-USPIO纳米粒悬液,经3.0T磁共振扫描仪获得T2*WI图像,测定各管T2*弛豫时间,评价所制备的脂质磁性纳米粒Gal-SLN-USPIO的负性强化效果；分别对摄取三种不同脂质磁性纳米粒后的人正常肝细胞L02和人肝癌细胞HepG2进行MR成像,以其信号强度的差异性分析不同脂质磁性纳米粒对L02和HepG2两种细胞的靶向摄取能力。研究结果表明,所制备的Gal-SLN-USPIO的T2*信号强度随浓度的增加而减低；T2*时间测量结果显示,随着脂质磁性纳米粒浓度的增高,T2*时间逐渐缩短,说明本研究所制备的Gal-SLN-USPIO具有负性造影效果,并存在一定的浓度效应关系,即在一定浓度范围内,Gal-SLN-USPIO浓度越高,其T2*WI负性强化效果越明显；体外细胞MR成像研究结果表明,Gal-SLN-USPIO被L02细胞摄取后,其信号强度降低最明显,所对应的R2*值明显大于SLN-USPIO和Gal-SLN-USPIO/PEG组(p0.05)；三种脂质磁性纳米粒被HepG2细胞摄取后,其对应的信号强度及R2*值三者之间无明显差异(p0.05),这一研究结果也与之前的体外细胞摄取研究结果相吻合。以上结果提示以Gal-SLN-USPIO作为对比剂所构建的MR探针具有应用于体内成像的可行性,为进一步地体内原位肝癌成像实验奠定了一定的基础。 第四部分：脂质磁性纳米粒的体内MR分子成像研究 本研究采用肝癌组织块原位移植法构建HepG2裸鼠原位移植肝癌模型；将肿瘤模型随机分为三组,每组裸鼠通过尾静脉分别注射三种脂质磁性纳米粒(剂量约含铁0.5μg/g体重)后,采用小动物线圈经3.0T磁共振(MR)扫描获得不同时间点的横断位T2WI图像,测量增强前及增强后不同时间点每组的正常肝组织、肿瘤及背景噪声的MR信号强度(SI),计算肝脏组织与肿瘤的信噪比(SNR)、肿瘤-肝脏的对比信噪比(CNR)、每组增强前后之间及增强后三组间CNR的差异；磁共振扫描结束后处死裸鼠,切取含瘤肝脏组织做常规HE染色,以证实肿瘤模型构建的成功与否；做普鲁士蓝染色,进一步证实不同脂质磁性纳米粒在肝脏中的靶向性分布情况。体内MR成像的研究结果表明,三组裸鼠增强后不同时间点T2WI序列肝内肿瘤的SNR较增强前均无显著性差异(p0.05),正常肝组织的SNR较增强前均有显著性差异(p0.05),增强后Gal-SLN-USPIO组肿瘤-肝脏的CNR较SLN-USPIO和Gal-SLN-USPIO/PEG组明显提高且有显著性差异(p0.05)；病理切片HE染色结果表明,本研究所构建的HepG2裸鼠原位移植肝癌模型全部接种成功；普鲁士蓝染色结果表明,Gal-SLN-USPIO组正常肝脏组织中的蓝色铁颗粒明显多于SLN-USPIO和Gal-SLN-USPIO/PEG组,进一步证实了Gal-SLN-USPIO对肝脏的靶向性明显强于SLN-USPIO和Gal-SLN-USPIO/PEG.以上研究结果提示Gal-SLN-USPIO有望成为肝脏靶向的特异性MR探针。 总结以上实验,本研究成功制备了对肝脏具有主被动双靶向功能的脂质磁性纳米粒Gal-SLN-USPIO,并通过体内外实验证实Gal-SLN-USPIO在肝脏靶向磁共振分子成像中具有良好的应用前景。
[Abstract]:Solid lipid nanoparticles (SLN) is a new type of nanoparticle drug delivery system which is developing in recent years. The solid or synthetic lipids such as glycerol monostearate, lecithin and three acyl glycerol are used as carriers to encapsulate drugs or contrast agents in the lipid nucleate into an acid state agent. Sex, after intravenous injection into the body, is quickly absorbed by the reticuloendothelial system (Reticuloendothelial system, RES) and passively enters the liver and spleen and has a significant passive liver targeting effect. The Asialoglycoprotein receptor (ASGPR) is a specific expression of the liver parenchyma cells that are completely expressed in the hepatic sinusoid gap of the mammalian liver. Membrane surface proteins can specifically identify glycoproteins with galactose residues. Each liver cell has as many as 5105 receptors on the surface of each liver, while in liver diseases such as hepatitis, liver cirrhosis, liver cancer or liver metastasis, the number and function are decreased. Galactose is a specific ligand for the ASGPR of the liver cell surface and is a liver targeting group. The adhesion properties of hepatocytes on the extracellular matrix scaffolds were improved by using SLN as a carrier, using the passive targeting characteristics of the liver of SLN and the active target modification of the liver, and preparing the lipid magnetic nanoparticles Gal-SLN-US with a double targeting function of the liver with the semi lactose modified SLN as the inclusion material. PIO. In vitro and in vivo, the target effect on normal liver cells, hepatoma cells and normal liver tissues, as well as the diagnostic value for orthotopic liver cancer model were investigated. The main contents of the study include the following four parts:
Part one: preparation and physicochemical properties of lipid magnetic nanoparticles
In this study, the chemical coupling reaction between lactose acid (LA) and stearamide (ODA) was used to prepare the galactose - stearamide (Gal-ODA). The chemical structure was confirmed by nuclear magnetic resonance (1H NMR) and Fu Liye transform infrared spectroscopy (FTIR). On this basis, the monoglyceride was used as the lipid material and the aqueous solvent diffusion method was used. The lipid magnetic nanoparticles (SLN-USPIO), active targeted lipid nanoparticles (Gal-SLN-USPIO/PEG) and lipid magnetic nanoparticles (Gal-SLN-USPIO) with dual targeted function were not prepared for the passive targeting of the liver, including SLN-USPIO and Gal-SLN-USPIO/PEG as the control group and the physical and chemical properties of three lipid nanoparticles. The particle size, surface potential and distribution of three different lipid nanoparticles were measured with particle size particle size and surface potential analyzer. The morphology of lipid nanoparticles was observed by transmission electron microscopy (TEM). The results showed that the number of three lipid nanoparticles prepared by this study was between 45~60nm, The surface potential is between -27 and -35mV, the polydispersity index is between 0.14 and 0.35. There is no significant difference between the three groups. The lipid nanoparticles are all quasi spherical, the size is uniform, the surface is smooth and complete, the particle dispersion is good, and there is no obvious aggregation after 2 weeks at normal temperature. The external magnetic field adsorption experiments confirm that the lipid nanoparticles are all in the lipid nanoparticles. The package has magnetic Fe304 nanoparticles.
The second part: the in vitro cell experiment of liposome magnetic nanoparticles and the targeting of normal liver tissues in vivo.
In this study, the lipid magnetic nanoparticles were labeled with fluorescein isothiocyanate (FITC), the mouse mononuclear / macrophage RAW264.7 was used as the passive target cell model, the human normal liver cell L02 was the active target cell model and the human hepatoma cell HepG2 was the disease cell model. The laser confocal microscope qualitative and flow cytometry were used for quantitative research. The uptake of three kinds of lipid nanoparticles by three kinds of cells was investigated. The toxicity of different lipid magnetic nanoparticles to three kinds of cells was investigated with four methyl azazolazolide reaction colorimetric assay (MTT test), and liposoluble DiR was used as a near infrared fluorescent dye to prepare DiR labeled magnetic lipid nanoparticles, and small animals were used in vivo. The distribution of different lipid magnetic nanoparticles in normal nude mice was observed by a fluorescent imager. The results showed that the uptake of SLN-USPIO in RAW264.7 cells was stronger than that of Gal-SLN-USPIO and Gal-SLN-USPIO/PEG. The uptake of Gal-SLN-USPIO in LO2 cells was stronger than that of SLN-USPIO and Gal-SLN-USPIO/PEG, and the uptake of three lipid nanoparticles by HepG2 cells was observed. The competitive uptake experiments of two cells in L02 and HepG2 showed that the uptake of L02 cells to Gal-SLN-USPIO was stronger than that of HepG2 cells to Gal-SLN-USPIO. The results provided a possibility for subsequent molecular imaging and diagnosis of HCC model, and three lipid nanoparticles at a concentration of 100. The survival rates of all kinds of cells were above 80%, indicating that the lipid magnetic nanoparticles prepared in this study have low toxicity and have reliable safety for clinical application. The results of the study on the distribution of liver in vivo show that the liver distribution of Gal-SLN-USPIO with dual target function at different time points is stronger than that of SLN-USPIO and SLN-USPIO. Gal-SLN-USPIO/PEG control group, the results suggest that dual-targeting Gal-SLN-USPIO may be a promising contrast agent for the diagnosis of liver diseases.
The third part: in vitro MR imaging of lipid magnetic nanoparticles.
A Gal-SLN-USPIO nanoparticle suspension with 7 tubes of 0,1,10,15,25,50100 micron g/ml was prepared. The T2*WI image was obtained by a 3.0T magnetic resonance scanner. The T2* relaxation time of each tube was measured and the negative strengthening effect of the lipid magnetic nanoparticles Gal-SLN-USPIO was evaluated. The human normal liver after three different lipid nanoparticles was taken respectively. The cell L02 and human hepatoma cell HepG2 were imaging MR, and the target uptake ability of different lipid nanoparticles to L02 and HepG2 two cells was analyzed with the difference of its signal intensity. The results showed that the T2* signal intensity of the prepared Gal-SLN-USPIO decreased with the increase of concentration; T2* time measurement results showed that with lipid magnetic nano The increase of grain concentration and T2* time gradually shortened, indicating that the Gal-SLN-USPIO prepared by this study had negative contrast effect, and there was a certain concentration effect relationship, that is, the higher the concentration of Gal-SLN-USPIO, the more obvious the negative strengthening effect of T2*WI was, the MR imaging study of extracellular cells showed that Gal-SLN-USPIO was fined by L02. After the uptake, the signal intensity decreased most obviously, and the corresponding R2* value was significantly greater than that of the SLN-USPIO and Gal-SLN-USPIO/PEG group (P0.05). There was no significant difference in the corresponding signal intensity and R2* value between the three lipid nanoparticles after the uptake of HepG2 cells (P0.05). The results of this study were also with the previous results of cell uptake studies in vitro. The above results suggest that the MR probe constructed with Gal-SLN-USPIO as a contrast agent has the feasibility of applying in vivo imaging, which lays a foundation for further imaging experiments in vivo in vivo liver cancer.
The fourth part: in vivo MR molecular imaging of lipid magnetic nanoparticles.
In this study, the orthotopic liver cancer model of HepG2 nude mice was constructed by orthotopic transplantation of hepatocellular carcinoma tissue. The tumor models were randomly divided into three groups. Each group of nude mice injected three kinds of lipid nanoparticles (with a dose of about 0.5 mu g/g) through the tail vein in each group. The small animal coils were scanned by 3.0T magnetic resonance (MR) to obtain the different time points. The MR signal intensity (SI) of the normal liver tissue, tumor and background noise (SI), the signal to noise ratio (SNR) of the liver tissue and the tumor, the contrast signal to noise ratio (CNR) of the tumor liver (CNR), the difference of CNR between each group before and after enhancement and the three groups after enhancement were measured, and the MRI scan was executed after the MRI scan. In nude mice, the tumor bearing liver tissue was harvested for routine HE staining to verify the success of the tumor model construction, and Prussian blue staining was used to further confirm the targeting distribution of different lipid nanoparticles in the liver. The results of MR imaging in the body showed that the SNR of the three groups of nude mice at different time points in the T2WI sequence was compared with that of the T2WI sequence. There was no significant difference before the enhancement (P0.05), and the SNR of normal liver tissue was significantly different (P0.05). The CNR of tumor liver in Gal-SLN-USPIO group was significantly higher than that in SLN-USPIO and Gal-SLN-USPIO/PEG group (P0.05). The HE staining results of pathological section showed that the orthotopic transplantation of HepG2 nude mice in this study was in situ. All the liver cancer models were inoculated successfully, and Prussian blue staining showed that the blue iron particles in the normal liver tissues of the Gal-SLN-USPIO group were obviously more than that of the SLN-USPIO and Gal-SLN-USPIO/PEG groups. It was further confirmed that the target of Gal-SLN-USPIO to the liver was significantly stronger than that of SLN-USPIO and Gal-SLN-USPIO/PEG., suggesting that Gal-SLN-USPIO has a Gal-SLN-USPIO. It is expected to be a specific MR probe for liver targeting.
In this study, the lipid magnetic nanoparticles Gal-SLN-USPIO, which has the primary and passive dual targeting function of the liver, has been successfully prepared, and it has been proved that Gal-SLN-USPIO has a good application prospect in the target magnetic resonance imaging of the liver in vivo and in vitro.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R943;R445.2
本文编号：2160848
[Abstract]:Solid lipid nanoparticles (SLN) is a new type of nanoparticle drug delivery system which is developing in recent years. The solid or synthetic lipids such as glycerol monostearate, lecithin and three acyl glycerol are used as carriers to encapsulate drugs or contrast agents in the lipid nucleate into an acid state agent. Sex, after intravenous injection into the body, is quickly absorbed by the reticuloendothelial system (Reticuloendothelial system, RES) and passively enters the liver and spleen and has a significant passive liver targeting effect. The Asialoglycoprotein receptor (ASGPR) is a specific expression of the liver parenchyma cells that are completely expressed in the hepatic sinusoid gap of the mammalian liver. Membrane surface proteins can specifically identify glycoproteins with galactose residues. Each liver cell has as many as 5105 receptors on the surface of each liver, while in liver diseases such as hepatitis, liver cirrhosis, liver cancer or liver metastasis, the number and function are decreased. Galactose is a specific ligand for the ASGPR of the liver cell surface and is a liver targeting group. The adhesion properties of hepatocytes on the extracellular matrix scaffolds were improved by using SLN as a carrier, using the passive targeting characteristics of the liver of SLN and the active target modification of the liver, and preparing the lipid magnetic nanoparticles Gal-SLN-US with a double targeting function of the liver with the semi lactose modified SLN as the inclusion material. PIO. In vitro and in vivo, the target effect on normal liver cells, hepatoma cells and normal liver tissues, as well as the diagnostic value for orthotopic liver cancer model were investigated. The main contents of the study include the following four parts:
Part one: preparation and physicochemical properties of lipid magnetic nanoparticles
In this study, the chemical coupling reaction between lactose acid (LA) and stearamide (ODA) was used to prepare the galactose - stearamide (Gal-ODA). The chemical structure was confirmed by nuclear magnetic resonance (1H NMR) and Fu Liye transform infrared spectroscopy (FTIR). On this basis, the monoglyceride was used as the lipid material and the aqueous solvent diffusion method was used. The lipid magnetic nanoparticles (SLN-USPIO), active targeted lipid nanoparticles (Gal-SLN-USPIO/PEG) and lipid magnetic nanoparticles (Gal-SLN-USPIO) with dual targeted function were not prepared for the passive targeting of the liver, including SLN-USPIO and Gal-SLN-USPIO/PEG as the control group and the physical and chemical properties of three lipid nanoparticles. The particle size, surface potential and distribution of three different lipid nanoparticles were measured with particle size particle size and surface potential analyzer. The morphology of lipid nanoparticles was observed by transmission electron microscopy (TEM). The results showed that the number of three lipid nanoparticles prepared by this study was between 45~60nm, The surface potential is between -27 and -35mV, the polydispersity index is between 0.14 and 0.35. There is no significant difference between the three groups. The lipid nanoparticles are all quasi spherical, the size is uniform, the surface is smooth and complete, the particle dispersion is good, and there is no obvious aggregation after 2 weeks at normal temperature. The external magnetic field adsorption experiments confirm that the lipid nanoparticles are all in the lipid nanoparticles. The package has magnetic Fe304 nanoparticles.
The second part: the in vitro cell experiment of liposome magnetic nanoparticles and the targeting of normal liver tissues in vivo.
In this study, the lipid magnetic nanoparticles were labeled with fluorescein isothiocyanate (FITC), the mouse mononuclear / macrophage RAW264.7 was used as the passive target cell model, the human normal liver cell L02 was the active target cell model and the human hepatoma cell HepG2 was the disease cell model. The laser confocal microscope qualitative and flow cytometry were used for quantitative research. The uptake of three kinds of lipid nanoparticles by three kinds of cells was investigated. The toxicity of different lipid magnetic nanoparticles to three kinds of cells was investigated with four methyl azazolazolide reaction colorimetric assay (MTT test), and liposoluble DiR was used as a near infrared fluorescent dye to prepare DiR labeled magnetic lipid nanoparticles, and small animals were used in vivo. The distribution of different lipid magnetic nanoparticles in normal nude mice was observed by a fluorescent imager. The results showed that the uptake of SLN-USPIO in RAW264.7 cells was stronger than that of Gal-SLN-USPIO and Gal-SLN-USPIO/PEG. The uptake of Gal-SLN-USPIO in LO2 cells was stronger than that of SLN-USPIO and Gal-SLN-USPIO/PEG, and the uptake of three lipid nanoparticles by HepG2 cells was observed. The competitive uptake experiments of two cells in L02 and HepG2 showed that the uptake of L02 cells to Gal-SLN-USPIO was stronger than that of HepG2 cells to Gal-SLN-USPIO. The results provided a possibility for subsequent molecular imaging and diagnosis of HCC model, and three lipid nanoparticles at a concentration of 100. The survival rates of all kinds of cells were above 80%, indicating that the lipid magnetic nanoparticles prepared in this study have low toxicity and have reliable safety for clinical application. The results of the study on the distribution of liver in vivo show that the liver distribution of Gal-SLN-USPIO with dual target function at different time points is stronger than that of SLN-USPIO and SLN-USPIO. Gal-SLN-USPIO/PEG control group, the results suggest that dual-targeting Gal-SLN-USPIO may be a promising contrast agent for the diagnosis of liver diseases.
The third part: in vitro MR imaging of lipid magnetic nanoparticles.
A Gal-SLN-USPIO nanoparticle suspension with 7 tubes of 0,1,10,15,25,50100 micron g/ml was prepared. The T2*WI image was obtained by a 3.0T magnetic resonance scanner. The T2* relaxation time of each tube was measured and the negative strengthening effect of the lipid magnetic nanoparticles Gal-SLN-USPIO was evaluated. The human normal liver after three different lipid nanoparticles was taken respectively. The cell L02 and human hepatoma cell HepG2 were imaging MR, and the target uptake ability of different lipid nanoparticles to L02 and HepG2 two cells was analyzed with the difference of its signal intensity. The results showed that the T2* signal intensity of the prepared Gal-SLN-USPIO decreased with the increase of concentration; T2* time measurement results showed that with lipid magnetic nano The increase of grain concentration and T2* time gradually shortened, indicating that the Gal-SLN-USPIO prepared by this study had negative contrast effect, and there was a certain concentration effect relationship, that is, the higher the concentration of Gal-SLN-USPIO, the more obvious the negative strengthening effect of T2*WI was, the MR imaging study of extracellular cells showed that Gal-SLN-USPIO was fined by L02. After the uptake, the signal intensity decreased most obviously, and the corresponding R2* value was significantly greater than that of the SLN-USPIO and Gal-SLN-USPIO/PEG group (P0.05). There was no significant difference in the corresponding signal intensity and R2* value between the three lipid nanoparticles after the uptake of HepG2 cells (P0.05). The results of this study were also with the previous results of cell uptake studies in vitro. The above results suggest that the MR probe constructed with Gal-SLN-USPIO as a contrast agent has the feasibility of applying in vivo imaging, which lays a foundation for further imaging experiments in vivo in vivo liver cancer.
The fourth part: in vivo MR molecular imaging of lipid magnetic nanoparticles.
In this study, the orthotopic liver cancer model of HepG2 nude mice was constructed by orthotopic transplantation of hepatocellular carcinoma tissue. The tumor models were randomly divided into three groups. Each group of nude mice injected three kinds of lipid nanoparticles (with a dose of about 0.5 mu g/g) through the tail vein in each group. The small animal coils were scanned by 3.0T magnetic resonance (MR) to obtain the different time points. The MR signal intensity (SI) of the normal liver tissue, tumor and background noise (SI), the signal to noise ratio (SNR) of the liver tissue and the tumor, the contrast signal to noise ratio (CNR) of the tumor liver (CNR), the difference of CNR between each group before and after enhancement and the three groups after enhancement were measured, and the MRI scan was executed after the MRI scan. In nude mice, the tumor bearing liver tissue was harvested for routine HE staining to verify the success of the tumor model construction, and Prussian blue staining was used to further confirm the targeting distribution of different lipid nanoparticles in the liver. The results of MR imaging in the body showed that the SNR of the three groups of nude mice at different time points in the T2WI sequence was compared with that of the T2WI sequence. There was no significant difference before the enhancement (P0.05), and the SNR of normal liver tissue was significantly different (P0.05). The CNR of tumor liver in Gal-SLN-USPIO group was significantly higher than that in SLN-USPIO and Gal-SLN-USPIO/PEG group (P0.05). The HE staining results of pathological section showed that the orthotopic transplantation of HepG2 nude mice in this study was in situ. All the liver cancer models were inoculated successfully, and Prussian blue staining showed that the blue iron particles in the normal liver tissues of the Gal-SLN-USPIO group were obviously more than that of the SLN-USPIO and Gal-SLN-USPIO/PEG groups. It was further confirmed that the target of Gal-SLN-USPIO to the liver was significantly stronger than that of SLN-USPIO and Gal-SLN-USPIO/PEG., suggesting that Gal-SLN-USPIO has a Gal-SLN-USPIO. It is expected to be a specific MR probe for liver targeting.
In this study, the lipid magnetic nanoparticles Gal-SLN-USPIO, which has the primary and passive dual targeting function of the liver, has been successfully prepared, and it has been proved that Gal-SLN-USPIO has a good application prospect in the target magnetic resonance imaging of the liver in vivo and in vitro.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R943;R445.2
【共引文献】
相关硕士学位论文 前2条
1 初菁菁;Markov决策模型在乳腺癌筛查卫生经济学评价中的应用[D];浙江大学;2014年
2 何石林;腹腔镜与开腹肝切除术治疗肝硬化肝癌的安全性及预后分析[D];浙江大学;2014年
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