聚多巴胺介导的不同表面修饰纳米粒的构建及其体内外生物学行为的研究

发布时间：2018-03-20 08:50

本文选题：纳米制剂　切入点：表面包衣　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：我们知道纳米粒子的表面性质是影响其体内外生物学行为的重要因素,深入的了解并进一步控制纳米粒子的表面性质对纳米制剂安全有效的应用至关重要。近年来,纳米制剂包括用于治疗的纳米药物递送系统和用于诊断的纳米诊断试剂如聚合物纳米粒、脂质体、量子点、纳米磷灰石和石墨烯等,在疾病的诊断和治疗中显示出了巨大的应用前景,然而目前这些纳米制剂的临床应用仍然受到自身毒性,易被调理,细胞摄取效率低及自身疏水性、溶血性等不良的生物相容性的限制。为了改善纳米制剂的自身限制,达到延长纳米制剂在体内的循环时间、提高细胞摄取效率、降低毒性、延缓释放、提高生物相容性等目的,我们提出了一种聚多巴胺(p D)介导的简单通用的表面修饰策略:即利用聚多巴胺的二次修饰性能简单有效地对纳米制剂表面进行不同性质的修饰以达到提高其生物相容性,降低自身毒性,改善其药代动力学行为及提高其与细胞相互作用能力等目的。本文我们首先成功的制备了粒度均一、稳定性好的载抗癌药物拉洛他赛(LTX)的PLGA纳米粒,并将其作为模型纳米粒,通过聚多巴胺的介导成功地将polyethylene glycol(PEG)、bovine serum albumin(BSA)和poly-L-lysine(PLL)三种表面修饰物修饰于模型纳米粒表面,赋予其新的表面性质。接着对载有拉洛他赛的PLGA NPs,p D-PLGA NPs,BSA-p D-PLGA NPs,PLL-p D-PLGA NPs和PEG-p D-PLGA NPs五种纳米粒进行了的粒径、粒径分布、zeta电位及载药量的测定并利用透射电镜(TEM),X射线光电子能谱(XPS),核磁(1H-NMR)及红外(FTIR)对纳米粒的表面修饰进行进一步的结构确证。修饰前后粒径和电位的变化以及TEM、XPS、1H-NMR和FTIR的表征可以充分地证明聚多巴胺介导的不同表面修饰纳米粒的成功构建。为了考察三种表面包衣(BSA,PEG,PLL)对纳米粒血清稳定性的影响,我们进行了血清稳定性实验。结果表明,聚多巴胺介导的BSA包衣和PEG包衣可以很好的维持纳米粒子在血清蛋白中的稳定性,而PLL包衣虽然所带正电荷密度并不大,但还是会一定程度上的吸附血清蛋白,使纳米粒子的粒径增大,粒径分布变宽。此外,体外释放研究表明,BSA包衣对拉洛他赛的释放具有一定的缓释作用。而PEG包衣和PLL包衣对拉洛他赛的释放都没有明显影响,这一结果提示我们,纳米粒表面的BSA或与其结构类似的蛋白质包衣可以一定程度上的延缓像紫杉烷一类高血浆蛋白结合率药物的释放。以人乳腺癌细胞MCF-7及人肺癌细胞A549为细胞模型,考察上述三种表面包衣对纳米粒抗肿瘤活性及被癌细胞摄取效率的影响。空白制剂的细胞生存性研究表明,BSA包衣具有良好的生物相容性和安全性。此外,当粒子浓度高于250μg/m L时,聚多巴胺介导的PLL包衣才显示出轻微的毒性,这可能是由于其表面低密度的正电荷被带负电的血浆蛋白补偿后导致其并没有呈现严重的毒性。而载拉洛他赛表面修饰纳米粒的体外抗肿瘤活性结果表明,聚多巴胺介导的PLL包衣能够明显提升纳米粒的抗肿瘤活性,在弱酸性条件下,其体外抗肿瘤活性优势更显著。摄取结果表明,尽管聚多巴胺介导的PLL包衣所带的表面电荷密度并不大,但是它对纳米粒摄取的促进作用仍然要明显强于其他表面带负电的包衣。当p H由中性变为弱酸性时,PLL包衣表面正电荷密度的增加将会有助于提高纳米粒在肿瘤弱酸性条件下的细胞摄取。这一提高的程度因细胞种类的不同而不同。这一结果也成就了PLL包衣在用于弱酸性环境下肿瘤细胞杀伤方面的优势。本文采用了UPLC-MS/MS法来测定血浆样品中LTX的浓度,以雄性SD大鼠为试验动物,静脉注射给药后,做药时曲线,计算出主要药动学参数,以此来评价并比较聚多巴胺介导的不同表面包衣对纳米粒大鼠体内药动学行为的影响。体内药动学结果表明,PEG修饰的纳米粒子具有最好的体内药动学行为及参数。BSA包衣的纳米粒因其可以阻止血浆蛋白的吸附,故可以一定程度上地延长纳米粒子在体内的循环时间,提高其药时曲线下面积AUC(0-∞)。虽然PLL包衣纳米粒表面带有正电荷,但它的AUC(0-∞)和t1/2z仍分别是溶液剂的1.5倍(2642.77±133.50μg/L·h vs 1701.01±53.90μg/L·h,p0.05)和1.6倍(11.51±3.34h vs7.23±0.48h,p0.05)。最后以雄性昆明小鼠为实验动物,Di R为荧光染料,考察研究静脉注射聚多巴胺介导的不同表面包衣对纳米粒在小鼠体内组织分布的影响。经尾静脉注射后,用活体成像仪测定小鼠体内组织分布。结果表明由于一部分PLL包衣的纳米粒在注射进入血液循环过程中被血细胞内化或与血管壁表面上的聚阴离子结合,所以其在各个器官分布的总和少于其他两种带负电的表面包衣纳米粒。另外,PEG修饰的纳米粒在肝脾中的分布没有像BSA包衣的纳米粒那么迅速,而是慢慢地逐渐增多,这印证了PEG的优势的同时也表明外源粒子最终都难逃被网状内皮系统调理吞噬的命运。
[Abstract]:We know that the surface properties of nanoparticles is an important factor affecting the in vivo biological behavior, surface properties and further in-depth understanding of the control of nanoparticles is critical to the application of nano preparation is safe and effective. In recent years, including the preparation for nano nano drug delivery system for the diagnosis and treatment of diagnostic reagents such as nano polymer nanoparticles, liposomes, quantum point, nano apatite and graphene, in the diagnosis and treatment of diseases in which shows a great prospect, but the current clinical application of these nanoparticles remain under their toxicity, easily conditioning, cellular uptake efficiency and its hydrophobicity, compatibility constraints and other adverse biological hemolysis. In order to improve nano preparation of the self limiting, achieve the nano preparation and the circulating time prolonged, improve cellular uptake efficiency, reduce toxicity, delayed release, To improve the biocompatibility, we propose a polydopamine (P D) mediated by the simple and general surface modification: modification is simple and effective for different properties of surface modified nano preparation with two properties of poly dopamine in order to improve its biocompatibility, reduce their toxicity, improve the drug the pharmacokinetic behavior and improve their ability to interact with cells. In this paper we first prepared with uniform size were successfully loaded, good stability of anticancer drugs (LTX) match he Lalo PLGA nanoparticles, and the nanoparticles as a model, the polydopamine mediated successfully polyethylene glycol (PEG). Bovine serum albumin (BSA) and poly-L-lysine (PLL) three kinds of surface modification on the model of the surface of the nanoparticles, with new surface properties. Then the game PLGA NPs with him at P, D-PLGA NPs, BSA-p D-PLGA N Ps PLL-p, D-PLGA NPs and PEG-p D-PLGA NPs five nanoparticles were particle size, particle size distribution, zeta potential and drug loading were determined by transmission electron microscopy (TEM), X ray photoelectron spectroscopy (XPS), nuclear magnetic resonance (1H-NMR) and infrared (FTIR) surface modification of nanoparticles was confirmed further the modified particle size and zeta potential of the TEM, XPS, 1H-NMR and FTIR characterization can fully prove the different surface modified nanoparticles of poly dopamine mediated constructed successfully. In order to study the three kinds of surface coating (BSA, PEG, PLL) effect on the stability of nanoparticles in serum, we tested serum stability. The results show that the poly dopamine mediated BSA coating and PEG coating can maintain the stability of nanoparticles in the serum protein in well, and although the PLL coating with positive charge density is small, but it will be at a certain level of serum protein adsorption, The particle size increases, the particle size distribution becomes wider. In addition, the in vitro release showed that the BSA coating has a good effect on the controlled release to release his game. Lalo PEG coating and PLL coating had no obvious influence on the release of the game he Lalo, this result suggests that the nano particle surface or its BSA the similar structure of protein coating can to some extent delay as taxane a high plasma protein binding rate of drug release. In A549 breast cancer cells MCF-7 and lung cancer cell models, study the three kinds of surface coating on nanoparticles and antitumor activity by cancer cell uptake. The influence of cell survival the blank preparation showed that BSA coating has good biocompatibility and biological safety. In addition, when the particle concentration is higher than 250 g/m L, PLL coated poly dopamine mediated showed slight toxicity, which may Is due to plasma protein positive charge on the surface of the low density compensation is negatively charged after the lead did not show serious toxicity. And the carrier surface modified nanoparticles match he Ralo anti-tumor activity in vitro. The results showed that PLL coated poly dopamine mediated nanoparticles can significantly enhance the antitumor activity under weak acidic conditions. The in vitro antitumor activity advantage is more significant. Uptake results showed that although PLL coated poly dopamine mediated by the surface charge density is small, but its promoting effect on the uptake of nanoparticles is still significantly stronger than other negatively charged surface coating. When p H from neutral to weak acidic, positive charge density PLL coating surface increase will help to improve the nanoparticles in tumor cell uptake under weakly acidic conditions. The increasing degree is different because of the different types of cells. This result also achieved PLL coating In the weak acidic environment for tumor cell killing advantages. The UPLC-MS/MS concentration was measured in plasma samples of LTX, male SD rats as experimental animal, intravenous injection, drug time curve, calculate the main pharmacokinetic parameters, different surface coating in order to evaluate and compare the poly dopamine mediated effects of dynamic behavior of drug nanoparticles in rats. In vivo pharmacokinetic results show that in vivo, PEG nanoparticles modified with in vivo pharmacokinetic behavior and the best parameters of.BSA coated nanoparticles because it can prevent plasma protein adsorption, so to a certain extent on the extension of nanoparticles in vivo circulation time. The area under the AUC curve to improve the drug (0- 2). Although the PLL coating the surface of the nanoparticles with positive charge, but it's AUC (0- 2) and t1/2z were 1.5 times solution (2642.77 + 133.50 g/L h vs 1701.01 + 53.90 g/L - H, P0.05) and 1.6 times (11.51 + 3.34h vs7.23 + 0.48h, P0.05). The last male Kunming mice as experimental animal, Di R fluorescent dye, study on intravenous injection of different surface coated poly dopamine mediated effects of nanoparticles in mice by tail vein injection distribution. After the determination of in vivo distribution in vivo imaging instrument. The results show that as part of the PLL coated nanoparticles in injection into the blood circulation in the process of being internalized or blood cells and vascular wall on the surface of poly anion binding, so the distribution of various organs in less than the sum of the other two surface coating of negatively charged nanoparticles. In addition, the distribution of nanoparticles modified by PEG in the liver and spleen were not so rapidly as BSA nanoparticles coated, but slowly increased gradually, which confirms the advantage of PEG also showed that the foreign particles are difficult to escape by network The endothelial system recuperates the fate of the phagocytosis.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R943

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