自荧光聚丙烯酰胺纳米颗粒的合成及其荧光成像应用

发布时间：2018-05-25 00:19

本文选题：聚丙烯酰胺纳米粒子 + 自荧光　；参考：《东南大学》2017年硕士论文

【摘要】：荧光成像技术被广泛用于医学诊断和生物学领域。合理选择波长是成像的关键。与可见光比较,处于“光学窗”的近红外光具有组织穿透力强、信噪比高等优点,可获得组织深处的结构和功能信息,因此常被用于组织成像。在荧光成像中常将荧光素与载体结合构建荧光探针,纳米材料是常用的载体。与其他纳米材料比较,聚丙烯酰胺纳米材料因具有良好的生物相容性、表面官能团易控等优点而成为荧光素的理想载体。构建荧光探针时,荧光素和载体的固有性质均可能受交联作用的影响。另外,如果将多种荧光素结合到同一载体上,空间位阻可能导致荧光淬灭。为了解决这些问题,本研究尝试制备一种具有自发荧光属性的聚丙烯酰胺纳米粒子。本研究中,我们首先制备了封装ε-聚赖氨酸的聚丙烯酰胺纳米粒子(PAAPLNPs),之后用戊二醛交联PAAPLNPs,制备了具有自发荧光的PAAPLNPs(fPAAPLNPs)。fPAAPLNPs是粒径均一、单分散的纳米粒子,平均粒径为16.04±2.05nm;表面电荷为16.4 mV;具有较强的吸水能力,亲水性好。光谱分析表明fPAAPLNPs具有明显的520 nm和732 nm发射峰。NaBH4还原反应和红外光谱分析表明,fPAAPLNPs的荧光属性主要与C=N和C=C键有关。为了探究fPAAPLNPs与细胞的相互作用及细胞成像效果,我们用fPAAPLNPs处理了 RAW264.7、HepG2、Hepa1-6三种细胞。结果表明fPAAPLNPs具有很好的细胞内化及成像效果。荧光显微镜成像结果表明,fPAAPLNPs处理的细胞胞内呈现显著的红色及绿色荧光。近红外荧光(NIRF)扫描结果表明,fPAAPLNPs处理的细胞在700 nm处具有较强的NIRF信号,在800nm处的NIRF信号较弱。流式细胞分析表明,细胞内荧光随培养液中fPAAPLNPs浓度及孵育时间的增加而增强;同一浓度处理相同时间,RAW264.7胞内荧光最强,其次为Hepa1-6,HepG2最弱。细胞内荧光强弱与细胞吞噬fPAAPLNPs的量有关。CCK-8法测得细胞的半数致死量LD50分别为:6 mg/mL(RAW264.7)、7.5 mg/mL(Hepa1-6)、9 mg/mL(HepG2),说明 fPAAPLNPs 具有良好的生物相容性。裸鼠体内的NIRF成像结果显示:fPAAPLNPs可在体内有效成像,信噪比高。fPAAPLNPs经尾静脉进入血液后,被动靶向富集到肝脏,少量透过血脑屏障,随后被代谢清除。fPAAPLNPs的剂量达126 mg/kg体重时,裸鼠的活力仍不受影响,表明fPAAPLNPs具有良好的体内生物相容性。裸鼠尾部皮下注射fPAAPLNPs,少量被吸收进入血液,随后被清除;其余fPAAPLNPs残留皮下,注射后24天,其荧光未衰减,表明fPAAPLNPs在体内具有良好的荧光稳定性。利用fPAAPLNPs还可监测肿瘤生长。
[Abstract]:Fluorescence imaging is widely used in medical diagnosis and biology. Reasonable wavelength selection is the key to imaging. Compared with the visible light, the near-infrared light in the "optical window" has the advantages of strong tissue penetration and high signal-to-noise ratio, so it can obtain the structure and function information in the depth of the tissue, so it is often used in tissue imaging. Fluorescent probe is often constructed by combining fluorescein with carrier in fluorescence imaging. Nanomaterials are commonly used as carriers. Compared with other nanomaterials, polyacrylamide nanomaterials are ideal carriers for fluorescein because of their good biocompatibility and easy control of surface functional groups. The intrinsic properties of fluorescein and carrier may be affected by crosslinking. In addition, if several fluoresceins are bound to the same carrier, the steric hindrance may lead to fluorescence quenching. In order to solve these problems, we try to prepare a polyacrylamide nanoparticles with autofluorescence properties. In this study, we first prepared polyacrylamide nanoparticles encapsulated with 蔚 -Poly (lysine), and then crosslinked PAAPLNPs with glutaraldehyde to prepare homogenous and monodisperse PAAPLNPs(fPAAPLNPs).fPAAPLNPs nanoparticles. The average particle size is 16.04 卤2.05 nm, the surface charge is 16.4 MV, and the average particle size is 16.04 卤2.05 nm. Spectroscopic analysis shows that fPAAPLNPs has obvious emission peaks of 520nm and 732nm. NaBH4 reduction reaction and infrared spectrum analysis show that the fluorescence properties of fPAAPLNPs are mainly related to Con N and C bonds. In order to investigate the interaction between fPAAPLNPs and cells and the effect of cell imaging, we treated three kinds of RAW264.7 HepG2 Hepa1-6 cells with fPAAPLNPs. The results show that fPAAPLNPs has a good effect of cell internalization and imaging. Fluorescence microscopy showed that the cells treated with fPAAPLNPs showed significant red and green fluorescence. The results of NIRFs scanning showed that the cells treated with fPAAPLNPs had strong NIRF signals at 700nm and weak NIRF signals at 800nm. Flow cytometry analysis showed that the intracellular fluorescence increased with the increase of fPAAPLNPs concentration and incubation time in the culture medium, and the intracellular fluorescence of RAW264.7 was the strongest at the same time, and the weakest was Hepa1-6 and HepG2. The intracellular fluorescence intensity was related to the amount of fPAAPLNPs phagocytosis. The half lethal dose (LD50) measured by CCK-8 method was: 1 / 6 mg / mL RAW264.7 + 7.5 mg / mL + 9 mg / mL ~ (-1) Hep G2 ~ (2), which indicated that fPAAPLNPs had good biocompatibility. The results of NIRF imaging in nude mice showed that: fPAAPLNPs could be effectively imaged in vivo. When high SNR. FPAAPLNPs entered the blood through the tail vein, they were passively targeted to the liver and passed through the blood-brain barrier in a small amount, and then were metabolically cleared by the dose of .fPAAPLNPs up to 126 mg/kg body weight. The activity of nude mice remains unaffected, indicating that fPAAPLNPs has good biocompatibility in vivo. After subcutaneous injection of fPAAPLNPs into the tail of nude mice, a small amount of fPAAPLNPs was absorbed into the blood and then removed, while the remaining fPAAPLNPs residues were subcutaneously injected, and the fluorescence of fPAAPLNPs was not attenuated 24 days after injection, indicating that fPAAPLNPs had good fluorescence stability in vivo. FPAAPLNPs can also be used to monitor tumor growth.
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O631.24;R318

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