Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy.
Department of Pharmacy-Drug Sciences, University of Bari "Aldo Moro", Bari, Italy.
J Transl Med. 2018 May 23;16(1):140. doi: 10.1186/s12967-018-1509-4.
Chronic respiratory diseases, whose one of the hallmarks is oxidative stress, are still incurable and need novel therapeutic tools and pharmaceutical agents. The phenolic compounds contained in grape are endowed with well-recognized anti-oxidant, anti-inflammatory, anti-cancer, and anti-aging activities. Considering that natural anti-oxidants, such as proanthocyanidins, have poor water solubility and oral bioavailability, we have developed a drug delivery system based on solid lipid nanoparticles (SLN), apt to encapsulate grape seed extract (GSE), containing proanthocyanidins.
Plain, 6-coumarin (6-Coum), DiR- and GSE-loaded SLN were produced with the melt-emulsion method. Physicochemical characterization of all prepared SLN was determined by photon correlation spectroscopy and laser Doppler anemometry. MTT assay (spectrophotometry) and propidium iodide (PI) assay (cytofluorimetry) were used to assess cell viability. Flow cytometry coupled with cell imaging was performed for assessing apoptosis and necrosis by Annexin V/7-AAD staining (plain SLE), cell internalization (6-Coum-SLN) and reactive oxygen species (ROS) production (SLN-GSE). NF-κB nuclear translocation was studied by immunofluorescence. In vivo bio-imaging was used to assess lung deposition and persistence of aerosolized DiR-loaded SLN.
Plain SLN were not cytotoxic when incubated with H441 airway epithelial cells, as judged by both PI and MTT assays as well as by apoptosis/necrosis evaluation. 6-Coum-loaded SLN were taken up by H441 cells in a dose-dependent fashion and persisted into cells at detectable levels up to 16 days. SLN were detected in mice lungs up to 6 days. SLN-GSE possessed 243 nm as mean diameter, were negatively charged, and stable in size at 37 °C in Simulated Lung Fluid up to 48 h and at 4 °C in double distilled water up to 2 months. The content of SLN in proanthocyanidins remained unvaried up to 2 months. GSE-loaded SLN determined a significant reduction in ROS production when added 24-72 h before the stimulation with hydrogen peroxide. Interestingly, while at 24 h free GSE determined a higher decrease of ROS production than SLN-GSE, the contrary was seen at 48 and 72 h. Similar results were observed for NF-κB nuclear translocation.
SLN are a biocompatible drug delivery system for natural anti-oxidants obtained from grape seed in a model of oxidative stress in airway epithelial cells. They feature stability and long-term persistence inside cells where they release proanthocyanidins. These results could pave the way to novel anti-oxidant and anti-inflammatory therapies for chronic respiratory diseases.
慢性呼吸道疾病的一个标志是氧化应激，目前仍然无法治愈，需要新的治疗工具和药物。葡萄中的酚类化合物具有公认的抗氧化、抗炎、抗癌和抗衰老作用。考虑到天然抗氧化剂，如原花青素，水溶性和口服生物利用度差，我们已经开发了一种基于固体脂质纳米粒（SLN）的药物递送系统，能够包封含有原花青素的葡萄籽提取物（GSE）。
采用熔融乳化法制备普通、6-香豆素（6-Coum）、DiR 和 GSE 负载的 SLN。通过光子相关光谱法和激光多普勒动度法测定所有制备的 SLN 的理化特性。MTT 测定法（分光光度法）和碘化丙啶（PI）测定法（细胞荧光法）用于评估细胞活力。通过 Annexin V/7-AAD 染色（普通 SLE）、细胞内化（6-Coum-SLN）和活性氧（ROS）产生（SLN-GSE）评估细胞凋亡和坏死，采用流式细胞术结合细胞成像。通过免疫荧光研究 NF-κB 核易位。通过体内生物成像评估雾化 DiR 负载的 SLN 在肺部的沉积和持久性。
普通 SLN 孵育 H441 气道上皮细胞时无细胞毒性，PI 和 MTT 测定以及凋亡/坏死评估均如此。6-Coum 负载的 SLN 以剂量依赖性方式被 H441 细胞摄取，并在长达 16 天的时间内持续存在于细胞中，检测到的水平可检测到。SLN 在小鼠肺部可检测到 6 天。SLN-GSE 的平均直径为 243nm，带负电荷，在 37°C 的模拟肺液中在 48 小时内和在 4°C 的双蒸水中在 2 个月内保持稳定。原花青素中 SLN 的含量在 2 个月内保持不变。在加入过氧化氢刺激前 24-72 小时添加 GSE 负载的 SLN 可显著减少 ROS 产生。有趣的是，虽然在 24 小时时游离 GSE 确定的 ROS 产生降低高于 SLN-GSE，但在 48 和 72 小时时则相反。NF-κB 核易位也观察到类似的结果。
SLN 是一种生物相容性的药物递送系统，可从葡萄籽中提取天然抗氧化剂，用于气道上皮细胞氧化应激模型。它们具有稳定性和在细胞内的长期持久性，在细胞内释放原花青素。这些结果为慢性呼吸道疾病的新型抗氧化和抗炎治疗铺平了道路。
