Antitumor effect of iRGD-modified liposomes containing conjugated linoleic acid–paclitaxel (CLA-PTX) on B16-F10 melanoma

Ruo Du,¹ Ting Zhong,¹ Wei-Qiang Zhang,¹ Ping Song,¹ Wen-Ding Song,¹ Yang Zhao,¹ Chao-Wang,¹ Yi-Qun Tang,³ Xuan Zhang,^1,2 Qiang Zhang<sup>1,2

1</sup>Department of Pharmaceutics, ²State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, ³Department of Clinical Pharmacy, China Pharmaceutical University, Nanjing, People’s Republic of China

Abstract: In the present study, we prepared a novel delivery system of iRGD (CRGDK/RGPD/EC)-modified sterically stabilized liposomes (SSLs) containing conjugated linoleic acid–paclitaxel (CLA-PTX). The anti-tumor effect of iRGD-SSL-CLA-PTX was investigated on B16-F10 melanoma in vitro and in vivo. The in vitro targeting effect of iRGD-modified SSLs was investigated in a real-time confocal microscopic analysis experiment. An endocytosis-inhibition assay was used to evaluate the endocytosis pathways of the iRGD-modified SSLs. In addition, the in vitro cellular uptake and in vitro cytotoxicity of iRGD-SSL-CLA-PTX were evaluated in B16-F10 melanoma cells. In vivo biodistribution and in vivo antitumor effects of iRGD-SSL-CLA-PTX were investigated in B16-F10 tumor-bearing mice. The induction of apoptosis by iRGD-SSL-CLA-PTX was evaluated in tumor-tissue sections. Real-time confocal microscopic analysis results indicated that the iRGD-modified SSLs internalized into B16-F10 cells faster than SSLs. The identified endocytosis pathway of iRGD-modified SSLs indicated that energy- and lipid raft-mediated endocytosis played a key role in the liposomes’ cellular uptake. The results of the cellular uptake experiment indicated that the increased cellular uptake of CLA-PTX in the iRGD-SSL-CLA-PTX-treated group was 1.9-, 2.4-, or 2.1-fold compared with that in the CLA-PTX group after a 2-, 4-, or 6-hour incubation, respectively. In the biodistribution test, the CLA-PTX level in tumor tissues from iRGD-SSL-CLA-PTX-treated mice at 1 hour (1.84±0.17 µg/g) and 4 hours (1.17±0.28 µg/g) was 2.3- and 2.0-fold higher than that of CLA-PTX solution at 1 hour (0.79±0.06 µg/g) and 4 hours (0.58±0.04 µg/g). The value of the area under the curve for the first 24 hours in the tumors of iRGD-SSL-CLA-PTX-treated mice was significantly higher than that in the SSL-CLA-PTX and CLA-PTX solution-treated groups (P<0.01). The in vivo antitumor results indicated that iRGD-SSL-CLA-PTX significantly inhibited the growth of B16-F10 tumors compared with the SSL-CLA-PTX or CLA-PTX solution-treatment groups (P<0.01). The results of tumor-cell apoptosis showed that tumors from the iRGD-SSL-CLA-PTX-treated group exhibited more advanced cell apoptosis compared with the control, CLA-PTX solution-, and SSL-CLA-PTX-treated groups. In conclusion, the antitumor effect of iRGD-SSL-CLA-PTX was confirmed on B16-F10 melanoma in vitro and in vivo.

Keywords: peptide-modified liposome, iRGD, CLA-PTX, antitumor effect, in vitro, in vivo

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