两种RGD肽修饰的新型阿霉素脂质体的体外表征

摘要/Abstract

摘要： 目的作为配体, 肽对于多种受体显示出良好的靶向性, 例如在肿瘤表面过度表达的整合素家族受体。本文主要研究和表征分别用精氨酸-甘氨酸-天冬氨酸 (RGD) 三肽和甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸 (GRGDS) 五肽修饰的载药脂质体。方法分别采用RGD和GRGDS对包载阿霉素的立体稳定脂质体(SSL-doxorubicin)进行修饰, 以制备RGD-SSL-doxorubicin和GRGDS-SSL-doxorubicin。在体外表征试验中, 测定了各种脂质体的包封率、粒径、Zeta电位和释放率, 采用SRB试验研究了各脂质体对卵巢癌细胞的细胞毒作用, 并应用流式细胞仪和共聚焦显微镜考察了肿瘤细胞对各脂质体包封的阿霉素的摄取情况。结果所有脂质体的包封率均在95%以上, 采用RGD或GRGDS进行的修饰并未影响长循环脂质体的包封率。各种脂质体的平均粒径在105.7 ± 3.5 nm 和130.5 ± 3.0 nm之间, Zeta电位在–3.3 ± 0.3和–6.1 ± 0.3 mV之间, 在模仿体内环境的释放介质 (含胎牛血清) 中, 12小时内约有2/5的阿霉素从脂质体中释放。与游离阿霉素相比, 修饰后的脂质体对肿瘤细胞的抑制率略有下降; 在研究对阿霉素摄取的流式细胞试验和共聚焦试验中, 也有类似现象出现。将各种脂质体分别加入肿瘤细胞后, 阿霉素主要分布于SKOV-3的细胞核。结论本研究成功制备了两种分别用精氨酸-甘氨酸-天冬氨酸 (RGD) 三肽和甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸 (GRGDS) 五肽修饰的阿霉素脂质体。体外表征结果显示, 该修饰不会显著改变立体稳定脂质体的性质。

关键词: 脂质体, 脂质体, 脂质体, RGD修饰的阿霉素长循环脂质体, RGD修饰的阿霉素长循环脂质体, RGD修饰的阿霉素长循环脂质体, GRGDS修饰的阿霉素长循环脂质体, GRGDS修饰的阿霉素长循环脂质体, GRGDS修饰的阿霉素长循环脂质体, 表征, 表征, 表征

Abstract:

Aim Peptides as ligands have shown the active targeting properties to the receptors like integrins, a family of receptors over-expressed in cancers. The present study was to develop and characterize two peptides modified drug-containing liposomes. Methods Argine-glycine-aspartic acid (RGD) tripeptide and glycine-argine-glycine-aspartic acid-serine (GRGDS) pentapeptide were used for modifications on the doxorubicin-loaded sterically stabilized liposomes (SSL-doxorubicin) for the liposome preparation, RGD-SSL-doxorubicin and GRGDS-SSL-doxorubicin, respectively. Characterizations were performed by measurements of the encapsulation efficiency, particle size and zeta potential, release rates in a simulated in vivo environment, and cytotoxicity to ovarian cancer cells. Cell uptake was investigated by flow cytometry and confocal microscopy methods. Results All encapsulation efficiencies of the liposomes were above 95%, and the modifications using RGD or GRGDS did not affect the final encapsulation efficiency. Average particle sizes of the liposomes were in the range between 105.7 ± 3.5 nm and 130.5 ± 3.0 nm, and zeta potential values were between –3.3 ± 0.3 and –6.1 ± 0.3 mV. Approximately 2/5 of doxorubicin was released from liposomes before 12 h in the simulated in vivo environment containing fetal bovine serum. Inhibitory rates to cancer cells of the modified liposomes were slightly lower as compared to free doxorubicin. Similar phenomena were observed in the uptake measured by flow cytometry and confocal assay. After uptake applying various formulations on the cancer cells, doxorubicin was mainly distributed in the nuclei of SKOV-3 cells. Conclusion Two new doxorubicin-contained liposomes were successfully prepared and modified with argine-glycine-aspartic acid (RGD) tripeptide and glycine-argine-glycine-aspartic acid-serine (GRGDS) pentapeptide. In vitro characterization indicated that modifications did not alter significantly the properties of the sterically stabilized liposomes.

Key words: Liposomes, Liposomes, RGD-SSL-doxorubicin, RGD-SSL-doxorubicin, GRGDS-SSL-doxorubicin, GRGDS-SSL-doxorubicin, Characterization, Characterization

中图分类号:

R945

Supporting: Foundation item: National Natural Science Foundation of China (Grant No. 30572261) and the 985 Projects (Phase II) of the State Key Laboratory of Natural and Biomimetic Drugs (Peking University, China).
^*Corresponding author. Tel./fax: 86-10-82802791

罗春蕾, 赵慧, 王坚成, 张烜, 吕万良, 张强^*. 两种RGD肽修饰的新型阿霉素脂质体的体外表征[J]. .

Chun-Lei Luo, Hui Zhao, Jian-Cheng Wang, Xuan Zhang, Wan-Liang Lu, Qiang Zhang^*. In vitro characterization of two new doxorubicin liposomes modified with argine-glycine-aspartic acid tripeptide or glycine-argine-glycine-aspartic acid-serine pentapeptide[J]. .

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