A novel comb-like derivative CPEG-g-cholesterol was prepared by the reaction of cholesteryl chloroformate with hydroxyl groups of CPEG. The TEM and SEM results showed that CPEG-cholesterol spontaneously aggregated vesicles with the membrane thickness of 4.27 +/- 0.48 nm. Compared with the vesicles formed by comb-like PEG (CPEG), the derivation of cholesteryl chloroformate increased the thickness of vesicle membrane and developed corrugations. The hydrophobic doxorubicin (Dox) was added into the solution of CPEG and CPEG-g-cholesterol to test their vesicle stability. The drug-loaded vesicles of CPEG-g-cholesterol still existed but those of CPEG disappeared, which indicated that stability of vesicles was enhanced by the derived cholesteryl. chloroformate. The vesicles were further cross-linked by the reaction between divinyl sulfone (DVS) and the hydroxy groups in the side chains of the CPEG and CPEG-g-cholesterol. Both cross-linked vesicles of CPEG and CPEG-g-cholesterol entrapped considerable hydrophobic Dox in the vesicles membrane. The spontaneous vesicles of CPEG-g-cholesterol and the crosslinked vesicles of CPEG and CPEG-g-cholesterol might have great potential as a cargo of the hydrophobic drug.