以透明质酸修饰的单壁碳纳米管为载体的肿瘤靶向造影剂的研究

发布时间：2018-06-06 01:07

本文选题：钆 + 单壁碳纳米管　；参考：《郑州大学》2014年硕士论文

【摘要】：近年来，恶性肿瘤的发病率逐年攀升，其早期诊断尤为重要。磁共振成像技术是临床肿瘤诊断的重要手段之一，目前，临床上所使用的大部分磁共振造影剂均存在弛豫效能低、特异性差、半衰期短和毒性大的缺点。因此，发展一种新型造影剂迫在眉睫。有国外文献报道，将Gd3+沉积到短单壁碳纳米管（SWCNTs）内可以极大提高其弛豫效能。另外，SWCNTs还具有多个结合位点，在结合药物分子的同时还可以结合靶向分子，这为构建肿瘤靶向造影剂提供了新的思路。透明质酸（HA)是一种具有良好生物相容性、生物可降解性的天然线性多糖，研究发现肿瘤细胞表面存在多种HA特异性受体，利用HA这些特性可将其作为靶头装载到SWCNTs表面构建肿瘤靶向造影剂，从而达到药物缓释和靶向运输的目的。本文以HA修饰的单壁碳纳米管为肿瘤靶向转运载体，将顺磁性小分子GdCl3通过物理吸附作用吸附到HA-SWCNTs载体上，构建肿瘤靶向性Gd/HA-SWCNTs造影剂。通过细胞摄取实验、体内外毒性实验和肿瘤磁共振成像实验对该造影剂系统进行了全面综合评价，结果显示Gd/HA-SWCNTs是一种高效、定向、缓释、低毒的核磁共振造影剂。具体内容如下： 1.肿瘤靶向HA-SWCNTs载体的合成及Gd/HA-SWCNTs造影剂的构建。首先将SWCNTs通过浓硫酸和浓硝酸进行羧基化反应生成SWCNT-COOH，然后将HA通过亚烷基二胺为连接臂和其进行共价相连制得HA-SWCNTs载体，通过物理吸附作用将Gd3+负载到所得载体上，经过透析即得Gd/HA-SWCNTs。通过透射电镜、紫外全波长扫描和傅里叶红外光谱对SWCNTs的共价修饰进行表征，通过热重分析对所得载体进行分析，确证共价修饰。通过不同的药物载体比例对载药量进行筛选。透射电镜、紫外光谱、红外光谱和热重分析结果共同验证了HA-SWCNTs的成功制备，其中有42.27%的HA与SWCNTs成功偶联；当表面活性剂为10mg/ml的大豆卵磷脂，探头超声时间为30min，药物载体比例为7:1时载药效果最好，载药量达到22.58%。 2.荧光显微镜法和细胞流式分析法考察MCF-7细胞对HA-SWCNTs载体的摄取能力。荧光显微镜法结果显示，6h时HA-SWCNTs组信号达到高亮，比同时间点SWCNTs组信号强；细胞流式分析结果分析可知在6h时，MCF-7细胞对HA-SWCNTs的摄取率高达97.9%，而对SWCNTs的摄取率仅为87.4%。两种方法均验证了细胞对HA-SWCNTs的高摄取能力。 3.SRB法和HE染色法分别考察所制备Gd/HA-SWCNTs的体内外毒性。SRB法结果显示，当Gd3+浓度达到50μg/ml时，细胞生存率仍在90%以上，，Gd/HA-SWCNTs未表现出明显的细胞毒性；HE染色结果表明，当按照Gd3+为0.72mg/kg的剂量给药时，Gd/HA-SWCNTs对各器官均没有毒性作用。 4.化学方法测定Gd/HA-SWCNTs在小鼠肿瘤部位的吸收和靶向参数，用7.0T小动物专用磁共振成像仪测定该造影剂的成像性能。结果显示：Gd/HA-SWCNTs在小鼠肿瘤部位的吸收比其他对照组高，其成像信号强度高于对照组，且在24h时仍然有信号，这些结果均说明了所制备的Gd/HA-SWCNTs磁共振造影剂不仅具有肿瘤靶向性且可以在体内长时间存留，解决了目前临床造影剂靶向性差、半衰期短的问题。
[Abstract]:In recent years, the incidence of malignant tumor is increasing year by year, and its early diagnosis is particularly important. Magnetic resonance imaging is one of the important means of clinical cancer diagnosis. At present, most of the magnetic resonance imaging agents used in clinical use have the disadvantages of low relaxation efficiency, poor specificity, short half-life and large toxicity. The agent is imminent.
It is reported in foreign literature that the deposition of Gd3+ into short single walled carbon nanotubes (SWCNTs) can greatly improve the relaxation efficiency. In addition, SWCNTs also has multiple binding sites, which can combine the drug molecules with the target molecules, which provides a new way of thinking for the construction of tumor targeting contrast agents.
Hyaluronic acid (HA) is a natural linear polysaccharide with good biocompatibility and biodegradability. It is found that there are a variety of HA specific receptors on the surface of tumor cells. Using HA, it can be used as a target on the surface of SWCNTs to construct a tumor targeting contrast agent, so as to achieve the purpose of drug release and target transport.
In this paper, a HA modified single walled carbon nanotube is used as a tumor targeting carrier, and the paramagnetic small molecule GdCl3 is adsorbed on the HA-SWCNTs carrier by physical adsorption and the target Gd/HA-SWCNTs contrast agent is constructed. Through the cell uptake experiment, in vivo and in vitro toxicity experiments and tumor magnetic resonance imaging experiments, the contrast agent system has been completed. Comprehensive evaluation showed that Gd/HA-SWCNTs is an efficient, directional, slow release, low toxicity MRI.
1. the synthesis of the tumor targeting HA-SWCNTs vector and the construction of the Gd/HA-SWCNTs contrast agent. First, the carboxylation of SWCNTs through concentrated sulfuric acid and concentrated nitric acid was used to produce SWCNT-COOH. Then, HA was covalent connected by the alkyl two amine as the connection arm to produce the HA-SWCNTs carrier, and Gd3+ was loaded onto the obtained carrier by the adsorption of physical properties. After dialysis, the covalent modification of SWCNTs was characterized by transmission electron microscope (TEM), UV full wavelength scanning and Fourier transform infrared spectroscopy (FTIR). The covalent modification was confirmed by thermogravimetric analysis. The drug loading was screened by the proportion of different drug carriers. Transmission electron microscope, ultraviolet spectrum, infrared spectrum and heat were carried out by Gd/HA-SWCNTs.. The results of the reanalysis verified the successful preparation of HA-SWCNTs, of which 42.27% of HA was successfully coupled with SWCNTs; when the surface active agent was 10mg/ml, the ultrasonic time of the probe was 30min and the drug carrier ratio was 7:1, and the drug loading reached 22.58%.
2. fluorescence microscopy and cell flow analysis were used to investigate the uptake ability of MCF-7 cells to HA-SWCNTs carriers. The results of fluorescence microscopy showed that the signal of HA-SWCNTs group was higher at 6h than in SWCNTs group at the same time point. The results of cell flow analysis showed that the uptake rate of MCF-7 cells to HA-SWCNTs was up to 97.9% at 6h, and to SWC. The uptake rate of NTs was only 87.4%.. The two methods verified the high uptake ability of HA-SWCNTs.
The 3.SRB method and HE staining method were used to investigate the toxicity of Gd/HA-SWCNTs in vivo and in vitro, and the results showed that when Gd3+ concentration reached 50 u g/ml, the cell survival rate was still above 90%, and Gd/HA-SWCNTs did not show obvious cytotoxicity. HE staining results showed that Gd/HA-SWCNTs was no longer than the dosage of Gd3+ for 0.72mg/ kg. Toxic effects.
4. chemical method was used to determine the absorption and target parameters of Gd/HA-SWCNTs in the tumor site of mice. The imaging performance of the contrast agent was measured by the 7.0T small animal special magnetic resonance imaging instrument. The results showed that the absorption of Gd/HA-SWCNTs in the tumor site of mice was higher than that of the other control groups, and the intensity of imaging signal was higher than that of the control group, and the signal was still signal at 24h. The results show that the Gd/HA-SWCNTs magnetic resonance contrast agent not only has the tumor targeting and can stay in the body for a long time, but also solves the problem of poor targeting and short half-life of the clinical contrast agent.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943

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