A　lipid　derivative　of　gemcitabine　(Gem),　cholesteryl-phosphonyl　gemcitabine　(CPNG)　was　synthesized　in　this　study.　The　amphiphilicity　of　CPNG　was　confirmed　using　a　Langmuir　monolayer　method.　Nanoassemblies　were　formed　when　the　mixture　of　CPNG　and　a　long-circulating　material,　CHS-PEG(1500)　(9:1,　mol/mol)　were　injected　into　water.　The　nanoassemblies　could　be　spherical　vesicles　according　to　the　transmission　electron　microscopic　images.　Their　mean　size　was　71.1　nm　and　the　zeta　potential　was　-17.6　mV.　CPNG　maintained　stable　in　the　weakly　acidic　and　neutral　environments　although　mouse　plasma　quickly　degraded　CPNG.　The　cytotoxicity　of　the　nanoassemblies　was　3-6　folds　of　Gem＇s　cytotoxicity　on　five　human　cancer　cell　lines　including　95C,　95D,　A549,　SW620,　PANC-1　probably　because　of　the　phosphonyl　substitution　and　amphiphilicity　of　CPNG.　CPNG　mainly　distributed　into　the　mononuclear　macrophage　system　(including　liver　and　spleen)　after　bolus　intravenous　administration　of　the　nanoassemblies　into　mice　though　the　expected　significant　long-circulating　effect　was　not　shown.　The　nanoassemblies　with　the　high　dose　of　CPNG　showed　the　statistically　higher　in　vivo　anticancer　effect　than　Gem.　This　study　indicates　that　the　N-substituted　lipid　derivative　of　Gem　and　the　true　long-circulating　function　are　necessary　for　preparing　a　successful　nanoassembly　of　Gem.　(C)　2014　Published　by　Elsevier　B.V.