Cyclic RGD peptide-modified liposomal drug delivery system: enhanced cellular uptake in vitro and improved pharmacokinetics in rats

Zhongya Chen,^1,2 Jiaxin Deng,^1,2 Yan Zhao,^1,2 Tao Tao<sup>1,2

1</sup>National Pharmaceutical Engineering Research Center, ²Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai, People's Republic of China

Background: Integrins α_vβ₃ and α_vβ₅, both of which specifically recognize the Arg-Gly-Asp (RGD) motif, are overexpressed on many solid tumors and in tumor neovasculature. Thus, coupling the RGD motif to the liposomal surface for achieving active targeting can be a promising strategy for the treatment of tumors.
Methods: Cyclo(Arg-Gly-Asp-D-Phe-Cys) (cRGD) was covalently coupled with the liposomal membrane surface, followed by coating with poly(ethylene glycol) (PEG) using the post-insertion technique. The coupling efficiency of cRGD was determined. Doxorubicin as a model anticancer drug was loaded into liposomes using an ammonium sulfate gradient method to investigate the encapsulation efficiency, cellular uptake by the integrin-overexpressing human glioma cell line U87MG in vitro, and pharmacokinetic properties in Sprague-Dawley rats.
Results: cRGD was conjugated to the liposomal surface by a thiol-maleimide coupling reaction. The coupling efficiency reached 98%. The encapsulation efficiency of doxorubicin in liposomes was more than 98%. The flow cytometry test result showed that cRGD-modified liposomes (RGD-DXRL-PEG) had higher cell uptake by U87MG cells, compared with nontargeted liposomes (DXRL-PEG). The cellular uptake was significantly inhibited in the presence of excess free cRGD. Both the targeted (t_1/2 = 24.10 hours) and non-targeted (t_1/2 = 25.32 hours) liposomes showed long circulating properties in rat plasma. The area under the curve of the targeted and nontargeted liposomes was 6.4-fold and 8.3-fold higher than that of doxorubicin solution, respectively.
Conclusion: This study indicates preferential targeting and long circulating properties for cRGD-modified liposomes in vivo, which could be used as a potential targeted liposomal drug delivery system to treat human glioma.

Keywords: drug targeting, doxorubicin, covalent coupling, sterically stabilized liposomes, human glioma, post-insertion