【作者】 牛国琴；

【导师】 陆伟跃；

【作者基本信息】 复旦大学 ， 药剂学， 2004， 博士

【摘要】 鉴于人鼠嵌合抗肿瘤细胞核单抗chTNT能特异性靶向肿瘤坏死区瘤细胞的核抗原—DNA-组蛋白复合物，作者希望尝试将chTNT作为靶向头基连接到空间稳定脂质体表面，制备成既具有较长血循环时间又能靶向肿瘤细胞核抗原的免疫脂质体，以期达到疗效不低于抗肿瘤细胞表面抗原抗体修饰的免疫脂质体，而又能靶向多种实体瘤细胞的目的。 本文首先从普通和稳定性脂质体膜材料制备出发，制备了蛋黄卵磷脂（EPC）、氢化卵磷脂（HEPC）、甲氧基聚乙二醇-磷脂酰乙醇胺或氢化磷脂酰乙醇胺（MPEG-EPE或MPEG-HEPE）以及抗体与脂质体的桥连剂—吡啶二硫丙酰基-聚乙二醇-磷脂酰乙醇胺或氢化磷脂酰乙醇胺（PDP-PEG-EPE或PDP-PEG-HEPE）及其中间体单氨基聚乙二醇（PEG-NH₂）。TLC、IR、HNMR证实所制备的膜材料均符合实验要求。 空间稳定免疫脂质体（chTNT-SLs或IgG-SLs）的制备是采用高压乳匀法先制备出含PDP-PEG-EPE的空间稳定脂质体（PDP-SLs），再通过二硫苏糖醇（DTT）将其还原为表面带巯基的SLs（HS-SLs），最后与衍生化后的抗体连接即得，其粒径分布均匀，平均粒径约为140nm，稍大于修饰前粒径。包载阿霉素（DOX）则在制得PDP-SLs后进行，继后再连接抗体。采用硫酸胺梯度法包载DOX时，膜组成为EPC／Chol／MPEG-EPE或再加PDP-PEG-EPE的脂质体，包载条件以50℃、30min为优；膜组成为HEPC／Chol／MPEG-HEPE或再加PDP-PEG-HEPE的脂质体，包载条件65℃、30min为优，且药脂比为1：5时，包封率可达95％以上。 空间稳定免疫脂质体的免疫活性是决定其能否主动靶向的关键。¹²⁵I标记法的检测结果表明：抗体连接效率为53.72％，脂质体表面抗体密度为75.23μgAb／μmol磷脂，处于较优的抗体密度范围内；chTNT和chTNT-SLs与Raji细胞的特异性结合率均约为5％，与其核抗原结合率约15％；chTNT、chTNT-SLs均能与¹²⁵I-chTNT竞争结合Raji细胞核抗原，提示二者均有相同的免疫结合位点。ELISA法测定结果表明：IgG-SLs免疫活性约为小鼠IgG的43.7％，chTNT-SLs的免疫活性约为chTNT的50％，提示采用硫醚键方式将抗体连接于脂质体表面PEG末端的方法能在一定程度上保留抗体的免疫活性；chTNT-SLs与Raji细胞、KB细胞核抗原均能特异性结合，提示其具有广谱抗肿瘤细胞核抗原的作用。 空间稳定免疫脂质体的粒径及所包载DOX的膜泄漏稳定性决定着其在体内的生物学行为，在血循环中滞留时间长短是决定其能否靶向实体瘤组织的关键。放置过程中粒径变化结果提示，空间稳定免疫脂质体的稳定性介于普通脂质体和空间稳定脂质体之间；DOX-SL-IgG在37℃的PBS和1％人血浆中振荡放置10h药物泄漏或释放率分别为20.7％和9.5％，提示DOX空间稳定免疫脂质体膜阻止DOX泄漏性能较好;’“，I标记chTNT一CLS和chT’NT一SLS在大鼠体内药动学检测结果表明，后者在血液中清除慢，滞留时间比前者长。HPLC一荧光检测法测定Dox一sLS、Dox一sL一IgG和Dox在大鼠体内药动学的结果表明，药动学行为均符合两室模型，但DOX脂质体的tl几均比DOX长得多，提示DOX空间稳定免疫脂质体具有血液长循环特性。 空间稳定免疫脂质体在实体瘤组织积聚程度是其能否发挥疗效的基础，影像学定位是了解其在靶位动态积聚程度和速度的一种较为直观的方法。将磁共振用顺磁性显像剂的主药成分札喷酸复合物（Gd一DTPA）包载于脂质体中，制备成Gd一SL一chTNT。通过对给药后不同时相点的荷SKOV3瘤裸鼠进行磁共振增强扫描，结果表明，Gd一SL一ch丁NT在瘤体部位强化特征不同于游离Gd一DTPA，表现为12h和24h的后续强化效应。提示:空间稳定免疫脂质体能在实体瘤积聚，且积聚是个渐进过程。 DOX一SL一chTNT在荷SKOV3瘤裸鼠体内的初步抑瘤试验结果表明:DOX一SL一ch飞NT的瘤体相对体积和瘤重与空白对照组相比，均有显著性差异（P<0.ol和p<0.05）;其体积和重量抑瘤率分别为41 .20%和42.34%，与接有非特异性小鼠杭体IgG的DOX一SL一IgG相比有统计学差异（P<0 .1）;与Dox给药后对体重影响相比，DOx一SL一chTNT和Dox一sL一IgG组体重均稳中有升。提示:在相同药物剂量条件下，杭肿瘤细胞核单抗修饰的空间稳定免疫脂质体的杭肿瘤效果明显好于普通非特异性抗体修饰的空间稳定免疫脂质体，但两者均能明显降低DOX引起的毒副作用。更多还原

【Abstract】 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.更多还原