低相对分子质量壳聚糖-乙酰半胱氨酸偶合物肾脏靶向递送系统的研究

Synthesis and Evaluation of Low Molecular Weight Chitosan-Acetylcysteine Conjugate for Renal Targeting Therapy

目的合成低相对分子质量壳聚糖-乙酰半胱氨酸(LMWC-NAC)偶合物,并且评价其在急性肾损伤模型动物体内的肾脏靶向性及疗效。方法以碳二亚胺/N-羟基琥珀酰亚胺为缩合剂,将乙酰半胱氨酸通过酰胺反应嫁接到低相对分子质量壳聚糖上,并且采用核磁共振进行结构确证。在体外细胞实验中,考察LMWC-NAC偶合物被肾小管上皮细胞(HK-2)摄取的能力以及参与摄取过程的megalin受体。此外,用荧光材料吲哚菁绿标记LMWC-NAC偶合物,并对其在裸鼠体内的分布情况进行考察。腹腔注射脂多糖(20 mg·kg^(-1))构建急性肾损伤动物模型,给药72 h后检测各组小鼠的血肌酐、血尿素氮、炎症因子(TNF-α和IL-1β)、氧化应激(MDA),HE染色观察肾脏组织的病理学改变。结果通过酰胺反应得到LMWC-NAC偶合物,体外实验证实其通过megalin受体的介导被HK-2细胞摄取,而体内分布实验表明,LMWC-NAC偶合物在肾脏组织具有较好的分布,AKI小鼠尾静脉注射LMWC-NAC偶合物后,血清尿素氮和肌酐得到显著改善(P<0.01),炎症因子和氧化应激指标也显著下降(P<0.01),减轻肾脏病理损伤。结论 LMWC-NAC偶合物对肾脏具有较好的靶向性和改善其功能的能力,表明其可用于急性肾损伤的治疗。

OBJECTIVE To synthesize low molecular weight chitosan-acetylcysteine （LMWC-NAC） conjugate and investigate its renal targeting profile and the rapeutic effects in model mice with acute kidney injury （AKI）. METHODS NAC was conjugated to LMWC by EDC/NHS reaction and the LMWC-NAC conjugate was identified by ^1H-NMR. The cellular uptake of LMWC-NAC conju- gate and megalin receptor involved in this process was investigated in vitro. In addition, the tissue distribution of ICG-labelled LMWC- NAC conjugate was investigated in nude mice. AKI were induced by LPS intraperitoneal injection （20 mg·kg^-1 ）. The parameters in- cluding Scr, BUN, inflammatory factors （TNF-α and IL-1β）, and oxidative stress （MDA） were determined and renal histology was observed. RESULTS LMWC-NAC conjugate was successfully synthesized by the amide interaction. The in vitro studies demonstrated that the uptake of LMWC-NAC conjugate was mediated by the megalin receptor on HK-2 cells, and the tissue distribution experiment indicated that LMWC-NAC conjugate was mainly accumulated in the kidney. LMWC-NAC conjugate significantly suppressed Scr, BUN, inflammatory factors and oxidative stress （P 〈 0. 01 ） and improved kidney injury. CONCLUSION LMWC-NAC conjugate showed good renal targeting profile and effect in recovering renal functions, which indicates the potential of LMWC-NAC conjugate as a safe and efficient drug delivery system for the treatment of AKI.