Navath Raghavendra S, Kurtoglu Yunus E, Wang Bing, Kannan Sujatha, Romero Robert, Kannan Rangaramanujam M
Department of Chemical Engineering and Material Science, and Biomedical Engineering, Wayne State University, Detroit, Michigan 48202, USA.
Bioconjug Chem. 2008 Dec;19(12):2446-55. doi: 10.1021/bc800342d.
N-Acetyl-L-cysteine (NAC) is an antioxidant and anti-inflammatory agent with significant potential in clinical applications including stroke and neuroinflammation. The drug shows high plasma binding upon IV administration, requiring high doses and associated side effects. Through the use of an appropriate delivery vehicle, the stability and efficacy of NAC can be significantly improved. Dendrimers are an emerging class of nanoscale drug delivery vehicles, which enable high drug payloads and intracellular delivery. Poly(amidoamine) (PAMAM) dendrimer-NAC conjugates having cleavable disulfide linkages are designed for intracellular delivery based on glutathione levels. We have successfully synthesized two conjugates with a cationic G4-NH(2) and an anionic G3.5-COOH PAMAM dendrimer with NAC payloads of 16 and 18 per dendrimer, respectively, as confirmed by (1)H NMR and MALDI-TOF analysis. NAC release from the conjugates at intracellular and extracellular glutathione (GSH) concentrations were evaluated by reverse phase HPLC (RP-HPLC) analysis, and approximately 70% of NAC payload was released within one hour at intracellular GSH concentrations (approximately 10 mM), whereas negligible NAC release was observed at extracellular GSH levels (2 microM). FITC-labeled conjugates showed that they enter cells rapidly and localize in the cytoplasm of lipopolysaccharide (LPS)-activated microglial cells (the target cells in vivo). The significantly improved efficacies of dendrimer-NAC conjugates in activated microglial cells was confirmed by measuring the nitrite inhibition in the cell culture medium, which is an indication of the antioxidative property of the drug. Both G4-NH(2) and G3.5-COOH conjugates showed significantly better nitrite inhibition both at 24 and 72 h compared to free NAC, by as much as a factor of 16. The results indicate that PAMAM dendrimer conjugates produce higher local NAC concentration inside the cells, with GSH-sensitive disulfide linker enabling efficient and rapid cellular release of the drug.
N-乙酰-L-半胱氨酸（NAC）是一种抗氧化剂和抗炎剂，在包括中风和神经炎症在内的临床应用中具有巨大潜力。该药物静脉给药后血浆结合率高，需要高剂量且伴有副作用。通过使用合适的给药载体，NAC的稳定性和疗效可得到显著提高。树枝状大分子是一类新兴的纳米级药物递送载体，能够实现高药物负载量和细胞内递送。基于谷胱甘肽水平设计了具有可裂解二硫键的聚（酰胺胺）（PAMAM）树枝状大分子-NAC缀合物用于细胞内递送。通过（1）H NMR和基质辅助激光解吸电离飞行时间（MALDI-TOF）分析证实，我们已成功合成了两种缀合物，一种是阳离子型G4-NH(2)，另一种是阴离子型G3.5-COOH PAMAM树枝状大分子，每个树枝状大分子的NAC负载量分别为16和18。通过反相高效液相色谱（RP-HPLC）分析评估了缀合物在细胞内和细胞外谷胱甘肽（GSH）浓度下NAC的释放情况，在细胞内GSH浓度（约10 mM）下，约70%的NAC负载量在一小时内释放，而在细胞外GSH水平（2 microM）下观察到的NAC释放可忽略不计。异硫氰酸荧光素（FITC）标记的缀合物表明它们能迅速进入细胞并定位于脂多糖（LPS）激活的小胶质细胞（体内的靶细胞）的细胞质中。通过测量细胞培养基中的亚硝酸盐抑制作用证实了树枝状大分子-NAC缀合物在激活的小胶质细胞中显著提高的疗效，这是该药物抗氧化特性的一个指标。与游离NAC相比，G4-NH(2)和G3.5-COOH缀合物在24小时和72小时时均显示出显著更好的亚硝酸盐抑制作用，高达16倍。结果表明，PAMAM树枝状大分子缀合物在细胞内产生更高的局部NAC浓度，谷胱甘肽敏感的二硫键连接体能够使药物在细胞内高效快速释放。
