Research Center for Pharmaceutical Nanotechnology, Biomedicine Institute, Tabriz University of Medical Sciences, Tabriz, Iran.
Cardiovascular Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Colloids Surf B Biointerfaces. 2018 Apr 1;164:299-307. doi: 10.1016/j.colsurfb.2018.01.046. Epub 2018 Feb 3.
Oxidative stress possesses a key role in the onset and development of cardiovascular diseases (CVDs), thus it can be an efficient target to tackle such ailment. Marrubiin, a bioactive diterpene, is a potent antioxidant against oxidative stress. Herein, we aimed to formulate marrubiin loaded solid lipid nanoparticles (SLNs) to improve its pharmacokinetics and bioavailability and also to investigate free drug and formulation's protective impact against intracellular reactive oxygen species (ROS) generation in HUVECs. Marrubiin-SLNs were formulated using hot homogenization/solidification method and then were subjected to physicochemical characterizations, i.e. size, zeta potential, morphology, polydispersity index (PDI), encapsulation efficiency (% EE), drug loading/content and physical stability assessments. MTT assay was performed to study the cytotoxicity of the intact and SLN incorporated marrubiin on HUVECs. Further, the antioxidant property of marrubiin and formulations was evaluated using DPPH radical scavenging assay and their protective effect against TNF-α induced oxidative stress was assessed by the means of intracellular ROS assessment, and also apoptosis/necrosis, cell cycle, and DNA fragmentation assays. Electron microscopy analysis showed spherical monodispersed SLNs with the size less than 100 nm, particle/zeta size analyses also approved the size of particles with a zeta potential of -1.28 ± 0.17 mV. Results also showed high EE (98%), drug loading (31.74 mg/g) with 3.15% drug content. In vitro release studies revealed about 90% of marrubiin cumulative release during 24 h. The stability of marrubiin-SLNs in terms of size, zeta potential, polydispersity index, EE and drug leakage was approved. Marrubiin antioxidant stability after formulation was approved by DPPH analysis. MTT cell survival assay showed no significant cytotoxicity after 24 h and 48 h. Intracellular ROS detection assay revealed that marrubiin and marrubiin-SLNs, play protective effect against TNF-α induced oxidative stress in HUVECs which was further approved by apoptosis assessment. Conclusively, based on our findings, marrubiin nanoparticles are proposed as a preventive/therapeutic remedy against disorders elicited by increased levels of intracellular ROS in CVDs.
氧化应激在心血管疾病 (CVDs) 的发生和发展中起着关键作用，因此它可以成为治疗这种疾病的有效靶点。Marrubiin 是一种生物活性二萜，是一种有效的抗氧化应激物质。在此，我们旨在将 marrubiin 制成负载固体脂质纳米粒 (SLNs)，以改善其药代动力学和生物利用度，并研究游离药物和制剂对 HUVECs 内活性氧 (ROS) 生成的保护作用。Marrubiin-SLNs 采用热匀化/固化法制备，然后进行理化特性评价，如粒径、Zeta 电位、形态、多分散指数 (PDI)、包封效率 (% EE)、载药量/含量和物理稳定性评估。MTT 法检测完整 marrubiin 和 SLN 包裹的 marrubiin 对 HUVECs 的细胞毒性。进一步采用 DPPH 自由基清除法评价 marrubiin 和制剂的抗氧化性能，并通过检测细胞内 ROS 评估 marrubiin 和制剂对 TNF-α 诱导的氧化应激的保护作用，以及通过细胞凋亡/坏死、细胞周期和 DNA 片段化分析评估。电子显微镜分析显示，SLNs 呈球形单分散，粒径小于 100nm，颗粒/Zeta 粒径分析也证实了粒径为-1.28±0.17mV 的颗粒。结果还显示，EE 为 98%，载药量为 31.74mg/g，药物含量为 3.15%。体外释放研究表明，24h 内 marrubiin 的累积释放率约为 90%。在尺寸、Zeta 电位、多分散指数、EE 和药物泄漏方面，证实了 marrubiin-SLNs 的稳定性。DPPH 分析证实了制剂后 marrubiin 的抗氧化稳定性。MTT 细胞存活率试验显示，24h 和 48h 后无明显细胞毒性。细胞内 ROS 检测显示，marrubiin 和 marrubiin-SLNs 对 TNF-α 诱导的 HUVECs 氧化应激具有保护作用，这一结果通过细胞凋亡评估得到进一步证实。综上所述，根据我们的研究结果，marrubiin 纳米粒可作为预防/治疗 CVDs 中细胞内 ROS 水平升高引起的疾病的药物。
