Abstract: Liposomes are of potential utility in vaccine as the carrier and adjuvant.The aim of this study was to prepare novel glutaryl cholesterol-liposomes which could be conjugated with the protein vaccine,and to study the storage and conjugation condition of liposomes.Glutaryl-cholesterol was synthesized using glutarate and cholesterol and identified by thin-layer chromatography (TLC),mass spectrometry (MS) and elemental analysis.Based on the synthesized glutaryl-chloesterol and phospholipids,glutaryl cholesterol-liposome was prepared using thin-film dispersion,and the formulation of the liposomes was optimized regarding the particle size and polydispersity index.The resultant glutaryl cholesterol-liposomes under the optimal condition have the particle size of 150-200 nm.In addition,the stability of the optimized glutaryl cholesterol-liposomes was observed under the selected temperature.It was shown that lyophilized glutaryl cholesterol-liposomes could be stable at -20 °C up to 12 months.Furthermore,the conditions for the liposomal conjugation with H22 cell vaccine were evaluated to maximize the conjugation efficiency.After aqueous reconstitution,the liposomes were activated for 10min by adding N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC),and then incubated for 3 h with N-hydroxy S-succinimide (S-NHS) and the vaccine.The H22 cell vaccine-conjugated liposome was obtained with the conjugation efficiency of 81.4%.Therefore,novel glutaryl cholesterol-liposomes with the relatively better stability and significant capability of conjugation with the protein vaccine were successfully prepared.