College of Pharmacy, Seoul National University, Seoul, Republic of Korea.
J Pharm Pharm Sci. 2012;15(3):467-82. doi: 10.18433/j3z312.
Long-term stable cationic solid lipid nanoparticles (cSLNs) were formulated to transfer SMAD3 antisense oligonucleotides (ASOs) into the cells to enhance the intracellular activity of the ASOs. The SMAD3 ASOs were designed to block the inflammatory processes linked to TGFβ/SMAD3 pathway.
The cSLN formulation was prepared by high-pressure homogenization method composed of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), dioleoylphosphoethanolamine (DOPE), Tween 20, and tricaprin as a solid lipid core (1:1:1:1.67, w/w). The size and the zeta potential of the prepared cSLNs were measured by light scattering. The cSLN/ASO complexes were generated and introduced into the murine macrophage cells. After the treatment of the complexes, the cellular uptake of the complexes was determined by flow cytometry and the intracellular activity of SMAD3 ASOs from the complexes was evaluated by western blotting of SMAD3. In addition, TGFβ1, an upstream molecule of TGFβ/SMAD3 pathway, was monitored by ELISA.
The nano-scale sized cSLNs were positively charged and physically stable at 4oC during the storage up to 24 months. The uptake efficiency of the cSLN/ASO complexes into macrophage cells was enhanced up to 80% without cytotoxicity. After the treatment of the cSLN/ASO complexes, SMAD3 as well as TGFβ1 was significantly suppressed based on the SMAD3 ASO activity in the macrophage cells. In addition, the cSLN/ASO complexes prevented the morphological change to dendritic shape in the activated macrophage cells.
These results suggest that the cSLNs have a potential to deliver the SMAD3 ASOs to intracellular compartments for the anti-inflammatory effect. The development of this strategy might lead to anti-inflammatory and anti-fibrotic therapies in immunological disorders.
制备长期稳定的阳离子固体脂质纳米粒（cSLN），将 SMAD3 反义寡核苷酸（ASO）递送至细胞内，以增强 ASO 的细胞内活性。SMAD3 ASO 旨在阻断与 TGFβ/SMAD3 通路相关的炎症过程。
采用高压匀质法制备 cSLN 制剂，由 1,2-二油酰基-3-三甲铵丙烷（DOTAP）、二油酰基磷脂酰乙醇胺（DOPE）、吐温 20 和三辛酸甘油酯组成固体脂质核（1:1:1:1.67，w/w）。通过光散射法测量制备的 cSLN 的粒径和 zeta 电位。生成 cSLN/ASO 复合物并引入小鼠巨噬细胞。复合物处理后，通过流式细胞术测定复合物的细胞摄取率，并通过 Western blot 测定复合物中 SMAD3 ASO 的细胞内活性。此外，通过 ELISA 监测 TGFβ/SMAD3 通路的上游分子 TGFβ1。
纳米级 cSLN 带正电荷，在 4°C 下储存 24 个月时物理稳定性良好。cSLN/ASO 复合物进入巨噬细胞的摄取效率提高了 80%，且无细胞毒性。用 cSLN/ASO 复合物处理后，巨噬细胞中的 SMAD3 以及 TGFβ1 明显受到抑制，这与 SMAD3 ASO 的活性有关。此外，cSLN/ASO 复合物还防止了激活的巨噬细胞发生树突状形态变化。
这些结果表明 cSLN 具有将 SMAD3 ASO 递送至细胞内区室以发挥抗炎作用的潜力。该策略的发展可能为免疫性疾病的抗炎和抗纤维化治疗提供新的途径。
