Targeting To Cancer Cell's Nuclear Of Sterically Stabilized Liposomes Covalently Coupled With ChTNT

Due to its specific targeting to the nuclear antigent-DNA-histone, within necrotic tumor cells, chimeric TNT was attached to the surface of sterically stablized liposomes (SLs) as a targeting group in order that the immunoliposomes with both long circulation time and active targeting to nuclear antigen were obtained. It was expected that the therapeutic effect of chTNT-SLs would be increased, compared to the immunoliposomes modified with monoclonal antibody with recognition to tumor cell's surface antigen, and would target to variety kinds of solid tumors.In this study, the lipid materials, such as egg phosphatidylcholine (EPC) or hydrogenated egg phosphatidylcholine (HEPC), methoxy polyethylene glycol-phosphatidylethanolamine (MPEG-EPE or MPEG-HEPE), for preparing either conventional liposomes (CLs) or SLs were synthesized first. The coupling agents, pyridyldithioproprionate-PEG-EPE/HEPE (PDP-PEG-EPE or PDP-PEG-HEPE), for the attachment of antibody to liposomes were also synthesized. The purity and physicochemical properties of all these materials were assayed and proved using TLC, IR and NMR methods to meet the demand for preparing liposomes.The unilamellar liposomes with controllable sizes were prepared using the above mentioned materials via high pressure homogenization method. chTNT-SLs or IgG-SLs were prepared following the procedures below: First, SLs containing PDP-PEG-EPE were prepared. Then, the dithiol-bond of PDP was reduced to -SH by dithiothreitol (DTT) and HS-SLs were thus obtained. Finally, the derivative chTNT or IgG (maleimidophenylbutyrate-chTNT or IgG, MPB-chTNT or MPB-IgG)was linked to the surface of SLs.The target recognition was the key factor to the in vivo initiative targeting effect of sterically stabilized immunoliposomes (SILs). In this study, 125I radiolabeling tests were carried out. The results showed that the coupling ratio of antibody to liposomes was 53.72%, and the antibody density was 75.23ug/umol lipid. The binding ratio of chTNT and chTNT-SLs to Raji cells were about 5%, and to nuclear antigen, 15%. Both chTNT and chTNT-SLs could competitively bind with nuclear antigen against 125I-chTNT, which suggested that they all have same binding site. ELISA studies confirmed that the immunoreactivities of IgG-SLs and chTNT-SLs was about 43.7% and 50%, respectively, of murine IgG and chTNT, which indicated that the linkage method of antibodies to the terminal of PEG of liposome surface via thioether bond could retain their recognition. chTNT-SLs could bind specificly to nuclear antigen of both Raji cells and KB cells, which indicated that chTNT-SLs can target to different tumor cells.Doxorubicin (DOX) loaded liposomes were prepared by ammonium sulfate gradient method. The preparation conditions were optimized using Star design method. When the lipid composition included EPC, Choi and MPEG-EPE (with or without PDP-PEG-EPE), the liposomes could be obtained at 50C for 30 mins. But if the EPC was replaced by HEPC, the preparing condition would be 65C for 30 mins. The entrapment efficiency might reach 95% when the drug/phospholipid ratio was 1:5. After Dox was entrapped in liposomes, the antibody was conjugated to the liposomes, if necessary.The physical stability of liposomes influences the in vivo biological behavior. The size changing of liposomes during restoration at 4 C was investigated, and SILs stay between CLs and SLs on size stability. When DOX-SL-IgG were suspended in either PBS or 1% human plasma at 37C for 10 hrs, the leakage ratio of DOX was both relatively low (20.7%, 9.5%), which indicated that liposomes membrane could contain stably entrapped drugs.The circulation time of SILs determines the targeting effect to the solid tumors. The pharmacokinetic studies of 125I-chTNT-CLs and 125I-chTNT-SLs showed that the latter had longer circulation time. The pharmacokinetic performance of DOX-SLs, DOX-SL-IgG and DOX were also studied using HPLC-fluorescent assay, and the results showed that the pharmacokinetic module of DOX entrapped liposomes met the two-chamber module.