叶酸靶向癌细胞纳米荧光探针的制备及其性能研究

发布时间：2018-11-29 11:18

【摘要】：粒径在100～200 nm的纳米粒子具有良好的增强性渗透及滞留(Enhanced permeation and retention effect, EPR)效应,有望解决目前癌症检测准确率低、边缘不清的问题,已成为癌症靶向检测定位和治疗的研究热点。研究表明,具有良好水溶性和相容性的右旋糖酐可以通过自组装的方法,获得粒径在100～200 nm的纳米粒子,但在此基础上进一步添加叶酸(Folic acid, FA)作为靶向分子,通过主动靶向与被动靶向结合以提高粒子靶向作用从而实现癌症检测定位的研究非常少。本论文通过自组装的方式,制备以叶酸为靶向分子、荧光标记的粒径为100～200 nm的右旋糖酐粒子(Dex-DCA-FI/FA NPs),使其作为纳米癌细胞探针以实现主动靶向与被动靶向的结合检测并对其性能进行研究。研究内容包括以下几个方面：1.利用疏水的脱氧胆酸(Deoxycholic acid, DCA)对右旋糖酐进行修饰,利用自组装的方法,并通过对初始浓度及去离子水滴加速度的优化,制得直径峰值167.5 nm且分布均一的脱氧胆酸修饰的右旋糖酐纳米粒子。但粒子的稳定性并不理想,需进行改进。2.在Dex-DCA的基础上增加氨基,通过自组装的方法,制得多种粒径在100～200nm的氨基化脱氧胆酸修饰右旋糖酐纳米粒子(Dex-DCA-NH2 NPs).由于氨基的静电排斥作用,Dex-DCA-NH2 NPs在溶液中的稳定性大幅提高,可以存放15天以上且在细胞培养液中4h内粒径仍在100～200 nm。3.在Dex-DCA-NH2的基础上修饰异硫氰酸荧光素(Fluorescein isothiocyanate, FITC)作为荧光标记物、叶酸作为靶向分子,通过自组装的方法,制得三种具有荧光且粒径在100～200 nm的叶酸靶向FITC标记的脱氧胆酸修饰右旋糖酐纳米粒子(Dex-DCA-FI/FA NPs),三种纳米粒子在溶液中的稳定性良好,可以存放14天以上,在细胞培养液中4h内粒径仍200 nm左右。4. Dex-DCA-FI200.5/FA 0.5 NPs(上标表示Dex-DCA-FI合成时FITC和氨基的投料比,下标分别表示Dex-DCA-FITC和Dex-DCA-FA的含量)对叶酸受体(FR)过量表达的FR(+)组和FR表达量较少的FR(-)组癌细胞具有一定的区分能力；Dex-DCA-FI 500.75/FA 0.25 NPs对FR(+)组和FR(-)组癌细胞具有明显的区分能力,满足检测要求。且两种纳米粒子在浓度较低时对MCF-7细胞、Hela细胞、A549细胞的细胞毒性都很小,并都具有良好的血液相容性,有望进一步进行动物体内实验或通过改变标记物或靶向分子进行性能的改良。
[Abstract]:Nanoparticles with particle size of 100 ~ 200 nm have good enhancement of permeability and retention of (Enhanced permeation and retention effect, EPR), which is expected to solve the problem of low accuracy and unclear edge of cancer detection. It has become a hot spot in the research of targeted detection, localization and treatment of cancer. The results showed that the dextran with good water solubility and compatibility could be obtained by self-assembly method, and the nanoparticles with diameter of 100 ~ 200 nm could be obtained, but on this basis, folate (Folic acid, FA) was added as the target molecule. There is very little research on how to improve the targeting of particles by combining active and passive targets to achieve cancer detection and localization. In this paper, folic acid was used as the target molecule and the fluorescent labeled dextran particle (Dex-DCA-FI/FA NPs),) was prepared by self-assembly. It can be used as a probe to detect the active and passive targeting of cancer cells and to study its properties. The research includes the following aspects: 1. The hydrophobic deoxycholic acid (Deoxycholic acid, DCA) was used to modify dextran, and the self-assembly method was used to optimize the initial concentration and deionized water droplet acceleration. Deoxycholic acid modified dextran nanoparticles with a peak diameter of 167.5 nm and uniform distribution were prepared. However, the stability of particles is not ideal, and need to be improved. 2. Based on the addition of amino groups to Dex-DCA, a variety of 100~200nm nanoparticles (Dex-DCA-NH2 NPs).) modified by aminodeoxycholic acid (100~200nm) were prepared by self-assembly. Due to the electrostatic repellent effect of amino groups, the stability of Dex-DCA-NH2 NPs in solution was greatly improved, and the particle size of Dex-DCA-NH2 NPs could be stored for more than 15 days and within 4 hours in the cell culture medium, and the particle size was still 100 ~ 200 nm.3.. Based on Dex-DCA-NH2, fluorescein isothiocyanate (Fluorescein isothiocyanate, FITC) was modified as a fluorescent marker and folic acid as a target molecule. Three kinds of folic acid-targeted folic acid modified dextran nanoparticles (Dex-DCA-FI/FA NPs),) with fluorescence and diameter of 100 ~ 200 nm were prepared. The stability of Dex-DCA-FI/FA NPs), nanoparticles in the solution was good, and they could be stored for more than 14 days. The particle size was still about 200 nm in the cell culture medium within 4 h. Dex-DCA-FI200.5/FA 0.5 NPs (superscript represents the feed ratio of FITC to amino groups in the synthesis of Dex-DCA-FI, The subscript indicates the content of Dex-DCA-FITC and Dex-DCA-FA, respectively. It can distinguish the cancer cells of the FR () group which the folic acid receptor (FR) overexpression and the FR (-) group with less FR expression. Dex-DCA-FI 500.75/FA 0.25 NPs can distinguish cancer cells from FR () group and FR (-) group. The cytotoxicity of the two nanoparticles to MCF-7 cells, Hela cells and A549 cells at low concentration was very small and had good blood compatibility. It is expected that further in vivo experiments in animals or improvement of performance by altering markers or targeting molecules.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TB383.1;O657.3

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