School of Pharmacy, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 11031, Taiwan.
Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, Chiyoda, Tokyo 101-8308, Japan.
J Food Drug Anal. 2018 Apr;26(2):869-878. doi: 10.1016/j.jfda.2017.09.002. Epub 2017 Nov 10.
The stability and bio-distribution of genes or drug complexes with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO, Pluronic F-68) polymeric micelles (PM) are essential for an effective nanosized PM delivery system. We used Förster resonance energy transfer (FRET) pairs with PM and measured the FRET ratio to assess the stability of PM in vitro and in vivo on the cornea. The FRET ratio reached a plateau at 0.8 with 3% PM. Differential scanning calorimetry measurement confirmed the complex formation of FRET pairs with PM. Confocal imaging with the fluorophores fluorescein isothiocyanate isomer I (FITC) and rhodamine B base (RhB) also showed the occurrence of FRET pairs in vitro. The fluorophores were mixed with 3% PM solution or the FITC-labeled PEO-PPO-PEO polymers (FITC-P) were mixed with RhB-labeled plasmids (RhB-DNA). In addition, the in vitro corneal permeation of FRET pair complexes with PM reached a 0.8 FRET ratio. One hour after eye drop administration, FRET pairs colocalized in the cytoplasm, and surrounded and entered the nuclei of cells in the cornea, and the polymers were located in the corneal epithelial layers, as detected through anti-PEG immunohistochemistry. Furthermore, fluorescence colocalization in the cytoplasm and cell nucleus of the corneal epithelium was confirmed in tissues where RhB or RhB-DNA complexed with FITC-P was found to accumulate. We demonstrate that at a concentration of 3%, PM can encapsulate FRET pairs or RhB-DNA and retain their integrity within the cornea 1 h after administration, suggesting the feasibility and stability of PEO-PPO-PEO polymers as a vehicle for drug delivery.
基因或药物复合物与聚（环氧乙烷）-聚（环氧丙烷）-聚（环氧乙烷）（PEO-PPO-PEO，Pluronic F-68）聚合物胶束（PM）的稳定性和生物分布对于有效的纳米 PM 递药系统至关重要。我们使用 PM 与荧光共振能量转移（FRET）对，并测量 FRET 比，以评估 PM 在体外和体内角膜上的稳定性。PM 浓度为 3%时，FRET 比达到 0.8 的平台值。差示扫描量热法测量证实了 FRET 对与 PM 的复合物形成。用荧光染料异硫氰酸荧光素（FITC）和罗丹明 B 碱（RhB）的共聚焦成像也显示了体外 FRET 对的发生。将荧光染料与 3% PM 溶液混合，或将 FITC 标记的 PEO-PPO-PEO 聚合物（FITC-P）与 RhB 标记的质粒（RhB-DNA）混合。此外，PM 与 FRET 对复合物的体外角膜渗透达到 0.8 的 FRET 比。滴眼后 1 小时，FRET 对在细胞质中聚集，并包围并进入角膜细胞的细胞核，聚合物位于角膜上皮层，通过抗聚乙二醇免疫组化检测到。此外，在 RhB 或 RhB-DNA 与 FITC-P 结合的组织中，证实了细胞质和角膜上皮细胞核中的荧光共定位。我们证明，在 3%的浓度下，PM 可以包裹 FRET 对或 RhB-DNA，并在给药后 1 小时内在角膜内保持其完整性，这表明 PEO-PPO-PEO 聚合物作为药物传递载体的可行性和稳定性。
