摘要
研究目的
本课题以固体脂质纳米粒(SLN)为载体,负载钆喷替酸葡甲胺(Gd-DTPA)而制备纳米化MR对比剂。通过纳米化MR对比剂直肠给药后,大肠壁吸收纳米化对比剂后于T1WI的MR上增强显示及有助于大肠癌的显示,以建立基于固体脂质体纳米粒的新型MR大肠成像方法。
材料与方法
1.主要以硬脂酸及Gd-DTPA为原料,采用乳化-溶剂挥发法合成Gd-DTPA-SLN、FITC-Gd-DTPA-SLN及SLN三种纳米粒,并对纳米粒进行检测、体外释放及体外MR成像。
2.基于纳米化对比剂的正常小鼠大肠MR成像方法的建立及组织细胞学定位30只正常小鼠随机分5组(Gd-DTPA-SLN、FITC-Gd-DTPA-SLN、SLN、Gd-DTPA及水),每组各6只。分别进行小鼠乙状结肠及直肠灌药前、保留灌肠中及灌药后20分钟MR扫描。测量每只动物灌药前后大肠壁的T1值及大肠壁与周围正常肌肉C及CNR。此外,选择2只正常小鼠进行静脉注射Gd-DTPA作为对照。最后处死动物,取正常大肠壁进行组织细胞学分析。
3.基于纳米化对比剂的大肠癌MR成像方法的初步应用2只大肠癌小鼠乙状结肠及直肠灌药(Gd-DTPA-SLN)前、保留灌肠中及灌药后20分钟MR扫描。然后处死动物,取正常大肠壁进行组织学分析。
结果
1.采用乳化-溶剂挥发法合成Gd-DTPA-SLN、FITC-Gd-DTPA-SLN及SLN三种纳米粒平均粒径40.8nm-300.8nm,Gd-DTPA-SLN及FITC-Gd-DTPA-SLN中Gd-DTPA包封率分别为55.8%和55%。Gd-DTPA-SLN纳米粒在pH7.4的PBS中1h释放约32%,8h才达到平衡。体外MR成像显示Gd-DTPA-SLN、FITC-Gd-DTPA-SLN中的Gd-DTPA与Gd-DTPA溶液中T1驰豫时间相当。
2.基于纳米化对比剂的正常小鼠大肠MR成像方法的建立及组织细胞学定位Gd-DTPA-SLN及FITC-Gd-DTPA-SLN纳米粒保留灌肠T1WI的MR可以获得与Gd-DTPA保留灌肠MR成像类似的明腔MR结肠成像。由Gd-DTPA-SLN纳米粒及FITC-Gd-DTPA-SLN纳米粒保留灌肠20分钟后T1WI的FLAIR的MR图像与保留灌肠前相比,所有小鼠大肠壁全层明显均匀强化,然而SLN纳米粒、Gd-DTPA及蒸馏水对照组小鼠大肠壁信号强度则无明显变化。与MR图像一致,Gd-DTPA-SLN纳米粒及FITC-Gd-DTPA-SLN纳米粒组大肠壁T1驰豫时间保留灌肠20分钟后较灌肠前明显降低,而大肠壁与周围正常肌肉C及CNR保留灌肠20分钟后较灌肠前明显升高。FITC-Gd-DTPA-SLN保留灌肠20分钟后大肠壁荧光显微镜及激光共聚焦荧光显微镜显示大肠壁各层细胞核外部分包括细胞浆及细胞外间隙内均匀分布的由FITC发射的高浓度绿荧光。Gd-DTPA-SLN、SLN、Gd-DTPA及蒸馏水组保留灌肠20分钟后大肠壁内未见荧光物质。对照组则未见上述MR及组织细胞学表现。
3.基于纳米化对比剂的大肠癌MR成像方法的初步应用Gd-DTPA-SLN保留灌肠中T1WI的MR成像可以获得肿瘤明腔MR结肠成像。Gd-DTPA-SLN保留灌肠20分钟后T1WI的MR成像检查显示正常肠壁明显均匀强化,而大肠癌病灶的主体部分强化程度较轻,病灶与正常肠壁对比鲜明。
结论
1.采用乳化-溶剂挥发法合成负载Gd-DTPA的固体脂质体纳米粒具备肠道易吸收、Gd-DTPA负载量大及包封率高、粒径适宜及不改变Gd-DTPA的高顺磁性等特点。
2.本研究发挥MR及纳米技术的优势而建立了基于纳米的新型大肠成像方法,其主要特点:经直肠给药,而不需要静脉用药;一次灌药后可以获得明腔大肠MR成像及大肠壁吸收增强成像;FITC-Gd-DTPA-SLN纳米粒即可用于MR成像,又可用于组织细胞学的定位;纳米颗粒进入细胞内而实现细胞水平的显像;可能实现大肠吸收功能的评价。
3.基于固体脂质体纳米粒的新型MR大肠成像方法在大肠癌中的初步应用表明该方法具有可行性并有助于肿瘤病变的显示。
4.基于纳米化对比剂MR大肠成像方法的建立还可能成为结肠定位释药系统的新型体内评价方法。
5.临床上所用的3T MR扫描仪配置一小鼠专用线圈完全可以应用小鼠腹盆腔成像,并可获得高质量的MR图像。
Purpose
We attempted to develop a novel nanoparticle-based magnetic resonance (MR) colonography technique, which enabled to generate contrast-enhanced MRI of the colonic walls and demonstrate the colorectal carcinoma via a transrectal administration of solid lipid nanoparticle (SLN) loaded with gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA).
Materials and Methods
1. We synthesized three different SLNs, including blank SLNs, Gd-DTPA-loaded SLNs (Gd-DTPA-SLNs), and Gd-DTPA-SLNs conjugated with FITC (FITC-Gd-DTPA-SLNs) by solvent diffusion method. Physicochemical properties of different SLNs were characterized by using Zetasizer, fluorescence spectrophotometer and MR. In vitro release study of Gd-DTPA was performed.
2. Validation of the feasibility of the novel concept—nanoparticle-based MR colonography in normal mice and histological confirmation of the ability of transrectally-infused SLNs infiltrating to the colonic walls in normal mice.
The sigmoid-rectal colons of 30 normal mice were locally infused with various SLNs and control agents via a transrectal enema approach. The SLNs and control agents included Gd-DTPA-SLNs (n=6), FITC-Gd-DTPA-SLNs (n=6), blank SLNs (n=6), Gd-DTPA (n=6), and water (n=6). In the first phase of the study to validate the feasibility of MR colonography, the mice in each group were imaged with colonic MR imaging using a series of T1-weighted fluid-attenuated inversion-recovery (FLAIR) sequences, which were performed before, during and 20 min after transrectal enema with different SLNs and control agents. Then, the quantitative analyses were performed. The quantitative criteria were T1 relaxation times of the colorectal wall, colorectal wall-to-surrounding muscle contrast (C) and colorectal wall-to-surrounding muscle contrast-to-noise ratio (CNR). MR colonography in 2 normal mice were performed with intravenous injection of Gd-DTPA. In the second phase of the study, all mice were scarified to histologically confirm the novel concept after MR examination.
3. Validation of the feasibility of the novel nanoparticles-based MR colonography in DMH-induced colorectal tumor model.
Two mice in DMH-induced colorectal tumor model were imaged with colonic MR imaging using a T1-weighted fluid-attenuated inversion-recovery (FLAIR) sequence, which were performed before, during and 20 min after transrectal enema with Gd-DTPA-SLNs. Then, the mice were scarified for histological examination.
Results
1. Gd-DTPA-SLNs, FITC-Gd-DTPA-SLNs and blank SLNs displayed a size distribution of 40.8-300.8 nm with high Gd-DTPA entrapment efficiency at 55%-55.8%. In vitro MR showed that the T1 relaxation times of Gd-DTPA-SLN and FITC-Gd-DTPA-SLN were similar to that of Gd-DTPA. 32% Gd-DTPA was released from Gd-DTPA-SLNs at PH 7.4 buffer in 1 h. The release o Gd-DTPA reached a balance in 8 h.
2. The bright lumen MR Colonography was obtained when a T1-weighted FLAIR sequence was performed during transrectal enema with Gd-DTPA-SLNs and FITC-Gd-DTPA-SLNs. T1-weighted FLAIR MRI showed bright enhancement of colorectal wall, with the decrease of T1 relaxation time and the increase of colorectal wall-to-surrounding muscle C and CNR in all mice 20 min after transrectal enema with Gd-DTPA-SLNs and FITC-Gd-DTPA-SLNs. Of the colonic tissues treated with FITC-Gd-DTPA-SLNs, confocal laser-scanning microscope and fluorescence microscope demonstrated highly-concentrated green fluorescence (due to FITC emission), which localized in both extracelluar spaces and cytoplasms at various layers of the colorectal walls, including mucosa, submucosa, tunica muscularis, and serosa. These histological and MRI findings were not visualized in the control animal groups.
3. The bright lumen MR Colonography showed the masses when a T1 -weighted FLAIR sequence was performed during transrectal enema with Gd-DTPA-SLNs. MR colonography showed marked homogeneous enhancement of normal colorectal walls and mild inhomogeneous enhancement of the mass. The signal intensity in the lesion was much lower than that in the colorectal walls. There was a great contrast between the tumor and surrounding colorectal walls.
Conclusion
1. SLNs loaded Gd-DTPA by solvent diffusion method exhibit intestinal absorbablity, a suitable particle size, high loading efficiency, stability of paramagnetic properties of Gd-DTPA.
2. This study establishes the "proofs-of-principle" of a novel imaging technique—nanoparticle-based MR colonography by combining the advantages of both MR technology and nanotechnology. The properties of the novel MR colonography include that (i) MR colonography is based on transrectal enema with SLNs loaded Gd-DTPA without introvenous administration; (ii) the bright lumen MR Colonography and the similar dark lumen MR Colonography were obtained after transrectal enema with SLNs loaded Gd-DTPA; (iii) a useful imaging tool is provided for basic science to precisely establish MRI and histological correlation by using the Gd-FITC-SLNs we have produced; (iv) nanoparticles can penetrate into the cells of the colorectal walls in large quantities and cellular imaging is obtainable; and (v) a functional MRI can be potentially developed to assess colorectal absorption.
3. Validation of the feasibility of the novel nanoparticles-based MR colonography in DMH-induced colorectal tumor model demonstrates that the visualization of colorectal carcinoma based on colorectal absorption of Gd-DTPA-carrying nanoparticles on MR colonography is possible.
4. Nanoparticle-based MR colonography can become a novel in vivo evaluation method of colon delivery of drug.
5. Abdominal magnetic resonance imaging in mice using a clinical 3T MR scanner with a small coil is feasible and provides images of high quality.
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