Department of pharmaceutics, Universite de Montreal, CP 6128, Succursale Centre-ville, Montréal, Québec, Canada H3C 3J7.
Int J Pharm. 2011 Jun 15;411(1-2):178-87. doi: 10.1016/j.ijpharm.2011.02.039. Epub 2011 Mar 31.
In our previous study, PEG-g-PLA nanoparticles were developed and characterized. The aim of the present work is to investigate the effect of PEG grafting density (% PEG inserted onto poly(d, l)-lactide, PLA backbone) on both physicochemical and biological properties (mainly plasma protein binding and in vitro macrophage uptake) of PEG-g-PLA NPs. Rhodamine B (RHO) loaded NPs were prepared from a 1:1 (wt/wt) blend of PLA and PEG-g-PLA copolymer of varying PEG grafting density (1, 7, or 20% mol/mol of lactic acid monomer) by an o/w emulsion solvent evaporation method. These NPs were characterized with regard to their morphology, size, surface charge, loading efficiency, and rhodamine release. The extent of protein adsorption to the surface of different NPs was qualitatively investigated by dynamic light scattering technique. Additionally, the in vitro macrophage uptake following incubation of RAW 264.7 cells with rhodamine loaded PEG-g-PLA and PLA particles was investigated by confocal laser scanning microscopy (CLSM). The amount of NPs phagocytosed following incubation of RAW 264.7 cells with different concentrations of rhodamine loaded PLA or pegylated NPs for 24h at 37 °C was also determined by fluorescence spectroscopy. ALL lyophilized NPs showed larger diameter in the range of 300-400 nm compared to freshly prepared NPs suspension indicating particle aggregation upon lyophilization. % EE of rhodamine was found to be between 10% and 68% wt/wt depending on PEG grafting density. The higher the grafting density of PEG over PLA backbone, the more the entrapment efficiency. All pegylated NPs showed low zeta potential (close to zero) values. In vitro release analysis revealed that rhodamine leaked from all nanoparticles at a very slow rate at physiological pH, thus making it suitable for both imaging and uptake studies with RAW 264.7 cells. All PEG-g-PLA NPs of different PEG grafting density were well tolerated and exhibited no toxicity to RAW 264.7 cells as seen by cell proliferation assays. Cellular uptake of NPs was mainly dependent on polymer type as well as PEG grafting density. Grafted copolymer NPs resulted in lower degree of macrophage uptake compared to PLA NPs in macrophages cell lines. The higher the PEG grafting density, the lower the uptake of NPs by macrophage cells. Minimum NPs uptake for all the investigated concentrations was achieved when the PEG grafting density was 7% mol/mol of lactic acid. When increasing the PEG grafting density in the nanoparticles above 7%, no significant reduction in NPs phagocytosis was achieved. Thus, this study shows that the optimal PEG density required for designing stealth PEG-g-PLA NPs suitable for drug delivery applications might vary from 4 to 7%.
在我们之前的研究中，开发并表征了 PEG-g-PLA 纳米粒子。本工作旨在研究 PEG 接枝密度（插入到聚（d，l）-丙交酯（PLA）主链上的 PEG 的百分比）对 PEG-g-PLA NPs 的理化性质和生物学性质（主要是血浆蛋白结合和体外巨噬细胞摄取）的影响。通过 o/w 乳液溶剂蒸发法，从 PLA 和 PEG-g-PLA 共聚物（PEG 接枝密度分别为 1、7 或 20%mol/mol 乳酸单体）的 1:1（wt/wt）混合物中制备了罗丹明 B（RHO）负载的 NPs。这些 NPs 的形态、大小、表面电荷、载药效率和罗丹明释放情况进行了表征。通过动态光散射技术定性研究了不同 NPs 表面蛋白质吸附的程度。此外，通过共聚焦激光扫描显微镜（CLSM）研究了 RAW 264.7 细胞与负载罗丹明的 PEG-g-PLA 和 PLA 颗粒孵育后体外巨噬细胞的摄取情况。通过荧光光谱法还测定了 RAW 264.7 细胞在 37°C 下孵育不同浓度负载罗丹明的 PLA 或聚乙二醇化 NPs 24h 后吞噬的 NPs 量。所有冻干 NPs 的直径均大于刚制备的 NPs 悬浮液的 300-400nm，表明冻干过程中粒子聚集。Rhodamine 的 %EE 为 10%-68%wt/wt，取决于 PEG 接枝密度。PLA 主链上的 PEG 接枝密度越高，包封效率越高。所有聚乙二醇化 NPs 的 Zeta 电位均接近零。体外释放分析表明，在生理 pH 下，所有纳米颗粒的罗丹明泄漏速度非常缓慢，因此非常适合 RAW 264.7 细胞的成像和摄取研究。不同 PEG 接枝密度的所有 PEG-g-PLA NPs 均具有良好的耐受性，对 RAW 264.7 细胞无毒性，通过细胞增殖试验可见。细胞摄取主要取决于聚合物类型和 PEG 接枝密度。与 PLA NPs 相比，接枝共聚物 NPs 在巨噬细胞系中的摄取程度较低。PEG 接枝密度越高，巨噬细胞摄取的 NPs 越少。在所研究的所有浓度下，NP 摄取量最低时，PEG 接枝密度为 7%mol/mol 乳酸。当纳米粒子中的 PEG 接枝密度增加到 7%以上时，对 NP 吞噬作用没有明显的降低。因此，本研究表明，用于药物输送应用的设计隐形 PEG-g-PLA NPs 的最佳 PEG 密度可能在 4 到 7%之间。
