【摘要】：固體脂質(zhì)納米粒(Solid lipid nanoparticles, SLN)是近年來正在發(fā)展的一種新型納米粒給藥系統(tǒng),以固態(tài)的天然或合成的類脂如單硬脂酸甘油酯、卵磷脂、三酰甘油等為載體,將藥物或造影劑包裹于類脂核中制成酸態(tài)給藥體。因其表面的強(qiáng)疏水性,靜脈注射進(jìn)入機(jī)體后,很快被網(wǎng)狀內(nèi)皮系統(tǒng)(Reticuloendothelial system, RES)吞噬而被動進(jìn)入肝脾,具有顯著的被動肝靶向作用。去唾液酸糖蛋白受體(Asialoglycoprotein receptor, ASGPR)是一種完全由哺乳動物肝竇狀隙的肝實(shí)質(zhì)細(xì)胞特異性表達(dá)的膜表面蛋白,能夠特異性地識別帶有半乳糖殘基的糖蛋白,每個(gè)肝細(xì)胞表面有多達(dá)5105個(gè)受體,而在肝炎、肝硬化、肝癌或肝轉(zhuǎn)移癌等肝臟疾病時(shí),其數(shù)量和功能均有所下降。半乳糖是肝細(xì)胞表面ASGPR的特異性配體,是肝靶向基團(tuán),具有誘導(dǎo)和提高肝細(xì)胞在細(xì)胞外基質(zhì)支架材料上的粘附性能。本研究選擇以SLN作為載體,利用SLN自身的肝臟被動靶向特征,結(jié)合肝臟的主動靶向修飾技術(shù),制備了經(jīng)半乳糖修飾的以SLN為包被材料的對肝具有雙靶向功能的脂質(zhì)磁性納米粒Gal-SLN-USPIO,并在體外和體內(nèi)實(shí)驗(yàn)中考察了其對正常肝細(xì)胞、肝癌細(xì)胞及正常肝組織的靶向作用,以及對原位移植肝癌模型的診斷價(jià)值。具體研究內(nèi)容主要包括以下四個(gè)部分： 第一部分：脂質(zhì)磁性納米粒的制備及理化性質(zhì)研究 本研究首先采用乳糖酸(LA)和硬脂胺(ODA)之間的化學(xué)耦合反應(yīng)制備半乳糖-硬脂胺嫁接物(Gal-ODA),采用核磁共振氫譜(1H NMR)和傅里葉變換紅外光譜(FTIR)對其化學(xué)結(jié)構(gòu)進(jìn)行確證；在此基礎(chǔ)上,以單甘脂為脂質(zhì)材料,采用水性溶劑擴(kuò)散法分別制備對肝臟具有被動靶向功能的脂質(zhì)磁性納米粒(SLN-USPIO)、主動靶向功能的脂質(zhì)磁性納米粒(Gal-SLN-USPIO/PEG)及雙重靶向功能的脂質(zhì)磁性納米粒(Gal-SLN-USPIO),其中SLN-USPIO和Gal-SLN-USPIO/PEG作為對照組,并對三種脂質(zhì)磁性納米粒的理化性質(zhì)進(jìn)行了評價(jià)。采用微粒粒度及表面電位分析儀測定三種不同的脂質(zhì)磁性納米粒的粒徑、表面電位及其分布,采用透射電鏡(TEM)觀察脂質(zhì)磁性納米粒的形態(tài)。研究結(jié)果表明,本研究所制備得到的三種脂質(zhì)磁性納米粒數(shù)均粒徑在45~60nm之間,表面電位在-27～-35mV之間,多分散指數(shù)在0.14～0.35之間,三者之間無顯著性差異；脂質(zhì)磁性納米粒均呈擬球形,大小均勻,表面平滑完整,顆粒分散性良好,常溫放置2周后未見明顯聚集現(xiàn)象；外加強(qiáng)磁場吸附實(shí)驗(yàn)考察證實(shí)脂質(zhì)納米粒中均包裹有磁性Fe304納米顆粒。 第二部分：脂質(zhì)磁性納米粒的體外細(xì)胞實(shí)驗(yàn)及在體正常肝組織靶向性研究 本研究采用異硫氰酸熒光素(FITC)標(biāo)記脂質(zhì)磁性納米粒；以小鼠單核/巨噬細(xì)胞RAW264.7為被動靶向細(xì)胞模型,人正常肝細(xì)胞L02為主動靶向細(xì)胞模型和人肝癌細(xì)胞HepG2為疾病細(xì)胞模型,采用激光共聚焦顯微鏡定性和流式細(xì)胞計(jì)數(shù)儀定量研究三種細(xì)胞對不同脂質(zhì)磁性納米粒的攝取情況；采用四甲基偶氮唑鹽微量酶反應(yīng)比色法(MTT實(shí)驗(yàn))考察不同的脂質(zhì)磁性納米粒分別對三種細(xì)胞的毒性；采用脂溶性DiR作為近紅外熒光染料,制備DiR標(biāo)記磁性脂質(zhì)納米粒,采用小動物活體熒光成像儀觀察不同的脂質(zhì)磁性納米粒在正常裸鼠肝臟的分布。研究結(jié)果表明：RAW264.7細(xì)胞對SLN-USPIO的攝取強(qiáng)于Gal-SLN-USPIO和Gal-SLN-USPIO/PEG, LO2細(xì)胞對Gal-SLN-USPIO的攝取強(qiáng)于SLN-USPIO和Gal-SLN-USPIO/PEG,而HepG2細(xì)胞對三種脂質(zhì)磁性納米粒的攝取無明顯差異性；L02和HepG2兩種細(xì)胞共孵育競爭性攝取實(shí)驗(yàn)表明,L02細(xì)胞對Gal-SLN-USPIO的攝取明顯強(qiáng)于HepG2細(xì)胞對Gal-SLN-USPIO的攝取,這一研究結(jié)果為后續(xù)的在體肝癌模型分子成像及診斷提供了可能；三種脂質(zhì)磁性納米粒在濃度達(dá)到100μg/ml時(shí),各種細(xì)胞的存活率均在80%以上,說明本研究所制備的脂質(zhì)磁性納米粒具有較低的毒性,對臨床應(yīng)用具有可靠的安全性；在體肝臟分布研究結(jié)果表明,對肝臟具有雙靶向功能的Gal-SLN-USPIO在不同時(shí)間點(diǎn)的肝臟分布均強(qiáng)于SLN-USPIO和Gal-SLN-USPIO/PEG對照組,該結(jié)果提示具有雙靶向功能的Gal-SLN-USPIO有望成為肝臟疾病診斷的磁共振造影劑。 第三部分：脂質(zhì)磁性納米粒的體外細(xì)胞MR成像研究 配制7管鐵濃度分別為0、1、10、15、25、50,100μg/ml的Gal-SLN-USPIO納米粒懸液,經(jīng)3.0T磁共振掃描儀獲得T2*WI圖像,測定各管T2*弛豫時(shí)間,評價(jià)所制備的脂質(zhì)磁性納米粒Gal-SLN-USPIO的負(fù)性強(qiáng)化效果；分別對攝取三種不同脂質(zhì)磁性納米粒后的人正常肝細(xì)胞L02和人肝癌細(xì)胞HepG2進(jìn)行MR成像,以其信號強(qiáng)度的差異性分析不同脂質(zhì)磁性納米粒對L02和HepG2兩種細(xì)胞的靶向攝取能力。研究結(jié)果表明,所制備的Gal-SLN-USPIO的T2*信號強(qiáng)度隨濃度的增加而減低；T2*時(shí)間測量結(jié)果顯示,隨著脂質(zhì)磁性納米粒濃度的增高,T2*時(shí)間逐漸縮短,說明本研究所制備的Gal-SLN-USPIO具有負(fù)性造影效果,并存在一定的濃度效應(yīng)關(guān)系,即在一定濃度范圍內(nèi),Gal-SLN-USPIO濃度越高,其T2*WI負(fù)性強(qiáng)化效果越明顯；體外細(xì)胞MR成像研究結(jié)果表明,Gal-SLN-USPIO被L02細(xì)胞攝取后,其信號強(qiáng)度降低最明顯,所對應(yīng)的R2*值明顯大于SLN-USPIO和Gal-SLN-USPIO/PEG組(p0.05)；三種脂質(zhì)磁性納米粒被HepG2細(xì)胞攝取后,其對應(yīng)的信號強(qiáng)度及R2*值三者之間無明顯差異(p0.05),這一研究結(jié)果也與之前的體外細(xì)胞攝取研究結(jié)果相吻合。以上結(jié)果提示以Gal-SLN-USPIO作為對比劑所構(gòu)建的MR探針具有應(yīng)用于體內(nèi)成像的可行性,為進(jìn)一步地體內(nèi)原位肝癌成像實(shí)驗(yàn)奠定了一定的基礎(chǔ)。 第四部分：脂質(zhì)磁性納米粒的體內(nèi)MR分子成像研究 本研究采用肝癌組織塊原位移植法構(gòu)建HepG2裸鼠原位移植肝癌模型；將腫瘤模型隨機(jī)分為三組,每組裸鼠通過尾靜脈分別注射三種脂質(zhì)磁性納米粒(劑量約含鐵0.5μg/g體重)后,采用小動物線圈經(jīng)3.0T磁共振(MR)掃描獲得不同時(shí)間點(diǎn)的橫斷位T2WI圖像,測量增強(qiáng)前及增強(qiáng)后不同時(shí)間點(diǎn)每組的正常肝組織、腫瘤及背景噪聲的MR信號強(qiáng)度(SI),計(jì)算肝臟組織與腫瘤的信噪比(SNR)、腫瘤-肝臟的對比信噪比(CNR)、每組增強(qiáng)前后之間及增強(qiáng)后三組間CNR的差異；磁共振掃描結(jié)束后處死裸鼠,切取含瘤肝臟組織做常規(guī)HE染色,以證實(shí)腫瘤模型構(gòu)建的成功與否；做普魯士藍(lán)染色,進(jìn)一步證實(shí)不同脂質(zhì)磁性納米粒在肝臟中的靶向性分布情況。體內(nèi)MR成像的研究結(jié)果表明,三組裸鼠增強(qiáng)后不同時(shí)間點(diǎn)T2WI序列肝內(nèi)腫瘤的SNR較增強(qiáng)前均無顯著性差異(p0.05),正常肝組織的SNR較增強(qiáng)前均有顯著性差異(p0.05),增強(qiáng)后Gal-SLN-USPIO組腫瘤-肝臟的CNR較SLN-USPIO和Gal-SLN-USPIO/PEG組明顯提高且有顯著性差異(p0.05)；病理切片HE染色結(jié)果表明,本研究所構(gòu)建的HepG2裸鼠原位移植肝癌模型全部接種成功；普魯士藍(lán)染色結(jié)果表明,Gal-SLN-USPIO組正常肝臟組織中的藍(lán)色鐵顆粒明顯多于SLN-USPIO和Gal-SLN-USPIO/PEG組,進(jìn)一步證實(shí)了Gal-SLN-USPIO對肝臟的靶向性明顯強(qiáng)于SLN-USPIO和Gal-SLN-USPIO/PEG.以上研究結(jié)果提示Gal-SLN-USPIO有望成為肝臟靶向的特異性MR探針。 總結(jié)以上實(shí)驗(yàn),本研究成功制備了對肝臟具有主被動雙靶向功能的脂質(zhì)磁性納米粒Gal-SLN-USPIO,并通過體內(nèi)外實(shí)驗(yàn)證實(shí)Gal-SLN-USPIO在肝臟靶向磁共振分子成像中具有良好的應(yīng)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R943;R445.2

【共引文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前2條

1 初菁菁;Markov決策模型在乳腺癌篩查衛(wèi)生經(jīng)濟(jì)學(xué)評價(jià)中的應(yīng)用[D];浙江大學(xué);2014年

2 何石林;腹腔鏡與開腹肝切除術(shù)治療肝硬化肝癌的安全性及預(yù)后分析[D];浙江大學(xué);2014年

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本文編號：2160848