新型两亲性糖聚肽肝癌靶向诊疗一体化纳米粒子的制备及体内外实验研究

通过氨基酸环内酸酐进行开环聚合及Click反应,制备得到新型两亲性糖聚肽——含半乳糖结构单元的聚(N^ε-苄氧羰基-L-赖氨酸)/聚(L-谷氨酸酯)嵌段聚合物,通过红外光谱、核磁共振氢谱对产物结构进行表征;采用透析法制备糖聚肽纳米粒子,对其临界胶束浓度、粒径分布、表面形貌、生物活性、细胞毒性等进行考察。随后,以糖聚肽为载体包载近红外荧光染料IR780制备诊疗一体化纳米粒子,并对其在肝癌HepG2细胞的靶向摄取、光热疗效及活体成像效果方面进行评价。结果显示:该实验成功制备出含半乳糖结构单元的聚(N^ε-苄氧羰基-L-赖氨酸)/聚(L-谷氨酸酯)嵌段聚合物;这种糖聚肽在水溶液中的浓度达到0.015 μg/mL时,便可以通过自组装形成粒径大约85 nm的球形粒子。细胞毒性实验(MTT)表明,该纳米粒子具有较好的生物安全性,当其浓度达到500 μg/mL时,HepG2与HUVEC细胞仍保持90%以上的生存率。流式细胞术实验结果表明,以糖聚肽为载体,可高效地将IR780传送到HepG2细胞内,具有肝癌靶向能力;体外光热治疗实验结果证实,在808 nm波长激光照射下,对靶向吞噬诊疗一体纳米粒子的HepG2肝癌细胞具有杀伤作用,而且光热治疗效果与IR780的浓度成正相关,当IR780浓度为10.0 μM时,其治疗效果优于5.0 μM时的治疗效果。小动物活体成像实验表明,该诊疗一体化纳米粒子能特异性地聚集在肿瘤部位,经尾静脉注射24 h后仍具有明显的荧光特性。综上可见,该实验合成新型两亲性糖聚肽肝癌靶向诊疗一体化纳米粒子,在肝癌的靶向诊断与治疗中具有广阔的应用前景。

The Preparation and Biological Study of Amphiphilic Glycopolypeptides as Liver-Targeted Theranostic Nanoparticles

In this work, amphiphilic glycopolypeptides were synthesized by a sequential ring opening polymerization of ε-carbobenzyloxy-L-lysine N-carboxyanhydride and glutamic acid based N-carboxyanhydride monomer. Galactosyl sugar units as targeting ligands were conjugated to the polypeptides block via an efficient click reaction. The chemical structures of the obtained glycopolypeptides were characterized by Fourier transform infrared spectroscopy and nuclear magnetic resonance analysis. Glycopolypeptides base nanoparticles were prepared by dialysis method in aqueous solution, followed by characterized with fluorometry, transmission electron microscopy and dynamic light scattering. The biological activity was demonstrated by selective lectin binding experiments. The cytotoxicity of the glycopolypeptides was investigated by MTT test. Furthermore, liver targeted theranostic nanoparticles were prepared by using glycopolypeptides acted as nano-cargo to load near infrared dyes IR780. The cellular uptake, photothermal therapy effect and in vivo tumor imaging were investigated by using fluorescence imaging, quantitative flow cytometry and in vivo imaging system. Results indicated that the amphiphilic glycopolypeptides were obtained via the combination of ring open polymerization and “click” reaction. Own to their amphiphilic property, glycopolypeptides self-assembled into spherical nanoparticles with about 85 nm in diameter in an aqueous medium once the concentration was over 0.015 μg/mL. MTT results revealed that the glycopolypeptides nanoparticles was nontoxic to HepG2 cells and HUVECs even the concentration was up to 500 μg/mL. Fluorescence microscope revealed the specific recognition between glycopolypeptides and ricin agglutinin. According to the flow cytometry results, glycopolypeptides nanoparticles could deliver IR780 into the HepG2 cells effectively. Photothermal therapy study revealed that higher doses of IR-780 killed more tumor cells after laser irradiation at 808 nm. In vivo NIRF imaging shown that the theranostics nanoparticles were mainly accumulated in the tumor mass, even after 24 h, strong flourescence signals were still detected from the tumor mass. The glycopolypeptides were demonstrated promising tumor targeting theranostic nanocarriers.