Yifei Huang,1,* Tan Yang,2,* Wendian Zhang,2 Yao Lu,2 Peng Ye,2 Guang Yang,2 Bin Li,2 Shibo Qi,2 Yong Liu,2 Xingxing He,3 Robert J Lee,4 Chuanrui Xu,2 Guangya Xiang2 1Department of Pharmacy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People’s Republic of China; 2School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People’s Republic of China; 3Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People’s Republic of China; 4Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH, USA*These authors contributed equally to this workAbstract: Instability of targeting ligand is a roadblock towards successful development of folate targeted liposomes. Folate ligands have been linked to polyethylene glycol (PEG) and cholesterol by an amide bond to form folate-CONH-PEG-CONH-Cholesterol (F-CONH-PEG-CONH-Chol), which is subject to hydrolysis. To increase the stability of folate ligands and promote the long circulation and targeting effects, we synthesized a chemically stable lipophilic folate derivative, folate-CONH-PEG-NH-Cholesterol (F-CONH-PEG-NH-Chol), where the amide bond was replaced by a C-N bond, to deliver liposomal doxorubicin (Dox). Its physical stability, cellular uptake, cellular toxicity, pharmacokinetics, distribution, anti-tumor efficacy, and cardiac toxicity were investigated. Our results indicate that F-CONH-PEG-NH-Chol conjugated liposomes are taken up selectively by folate receptor-positive HeLa and KB cells. Compared with F-CONH-PEG-CONH-Chol with two carbonate linkages, F-CONH-PEG-NH-Chol better retained its drug entrapment efficiency and folate receptor-targeting activity during prolonged circulation. F-CONH-PEG-NH-Chol thus represents a physically stable and effective ligand for delivering folate receptor-targeted liposomes, with prolonged circulation time and efficient tissue distribution, as well as higher efficacy and less cardiac toxicity. Collectively, these results suggest that this novel conjugate can serve as a promising derivative for the delivery of anti-tumor therapeutic agents.Keywords: liposome, doxorubicin, folate ligand, FR-targeting, stability