【作者】 潘弘；

【导师】 陆伟跃； 周光兴； 姚明； 钟高仁； 刘敏；

【作者基本信息】 复旦大学 ， 药剂学， 2006， 博士

【摘要】 脂质体是一种由磷脂双分子层构成的具有水相内核的脂质微囊。通过表面修饰可以改变脂质体的性质和生物学行为：在脂质体表面修饰亲水性高分子（如聚乙二醇）可以阻碍血浆成分的调理作用，减少脂质体被网状内皮系统（RES）识别和摄取，使脂质体具有长循环的特点；在脂质体表面修饰特异性的配基（如抗体，多肽），能使脂质体具有主动靶向的特性。 人鼠嵌合抗肿瘤细胞核单抗（chemic TNT-3 monoclonal antibody，chTNT-3）是一种能特异性亲和肿瘤坏死区细胞核抗原的新型抗体。chTNT-3可特异性结合肿瘤坏死区暴露的单链DNA，定位在肿瘤坏死部位。由于实体瘤内部坏死区域的存在相当普遍，chTNT-3突破了常规肿瘤细胞单抗的局限，具有广谱抗肿瘤的特点。 本文选择chTNT-3作为靶向头基，制备了两种chTNT-3介导的主动靶向脂质体给药系统，并比较两种不同方式chTNT-3介导的主动靶向脂质体对肿瘤的靶向性：（1）空间稳定免疫脂质体通过脂质体表面的chTNT-3与肿瘤坏死部位的特异性结合作用靶向至肿瘤；（2）将生物素化chTNT-3（chTNT-3/B）预定位于荷瘤裸鼠的肿瘤坏死部位，再给予表面修饰链霉亲和素的空间稳定脂质体，通过生物素与链霉亲和素之间特异性结合作用将脂质体靶向至肿瘤。以阿霉素为模型药物，考察chTNT-3介导的主动靶向脂质体对荷瘤裸鼠的药效。 首先合成了脂质体空间稳定膜材料甲氧基聚乙二醇-氢化大豆磷脂酰乙醇胺（mPEG-HSPE）和用以连接抗体的功能性膜材料吡啶二硫丙酰胺-聚乙二醇-氢化大豆磷脂酰乙醇胺（PDP-PEG-HSPE），TLC、IR、¹H-NMR等初步鉴定为目的产物，符合试验要求。 主动靶向脂质体的制备包括两方面：（1）采用高压均质机或微型挤出器制备粒径均一的含PDP-PEG-HSPE的空间稳定脂质体（PDP-SL），通过硫酸铵梯度法包载模型药物阿霉素。以阿霉素包封率和载药量为指标，通过三因素五水平的星点设计-效应面优化法优化处方，得到较优的脂质体处方为：膜材料摩尔比为HSPC/CHOL/mPEG-HSPE/PDP-PEG-HSPE=5：4：0.2：0.05，药脂比0.18：1（w/w），包载温度65℃，包载时间20min时，包封率可达（93.2±2.0）％。（2）PDP-SL经二硫苏糖醇还原，得表面带巯基的脂质体（HS-SL），与马来酰亚胺苯基丁酰基chTNT-3（MPB-chTNT-3）连接得到空间稳定免疫脂质体（chTNT-3-SL）。另一方面，HS-SL与马来酰亚胺苯基丁酰基链霉亲和素（MPB-SAv）连接可得预定位脂质体（SAv-SL）；chTNT-3经生物素衍生化得到生物素取代度为3～8的chTNT-3/B，由chTNT-3/B和SAv-SL组成预定位脂质体给药系统。连接单抗或链霉亲和素后，chTNT-3-SL和SAv-SL的平均粒径分别从PDP-SL的102nm增大到123nm和114nm，脂质体表面抗体密度或链霉亲和素蛋白密度为1089μg/μmol PL和51.5μg/μmol PL，连接效率分别为69.8％和82.6％。建立相应ELISA法，测定chTNT-3衍生物的免疫活性，结果表明chTNT-3衍生化后的免疫活性几乎完全保留；chTNT-3通过共价方式连接到脂质体表面后，仍然在一定程度上保留了抗体的免疫活性。初步稳定性试验表明，chTNT-3-SL和SAv-SL在4℃贮存14d，两者的平均粒径及分布变化小，药物泄漏少于3％，chTHT-3-SL的相对免疫活性保持不变，理化性质较稳定。 为验证主动靶向脂质体的体外靶向性，建立了模拟肿瘤坏死部位的固定Raji细胞模型，制备包载钙黄绿素的主动靶向脂质体，利用荧光分析法验证空间稳定免疫脂质体和预定位脂质体给药系统与固定Raji细胞的特异性结合作用。结果表明，两种主动靶向脂质体给药系统与固定Raji细胞的结合量均显著高于阴性对照组（P＜0.001）。 以阿霉素为模型药物，制备了阿霉素空间稳定免疫脂质体（chTNT-3-SL[DXR]）和预定位脂质体给药系统（chTNT-3/B+SAv-SL[DXR]）。HPLC法测定了大鼠单剂量静注两种主动靶向脂质体及对照组空间稳定脂质体后阿霉素在大鼠体内的药动学参数，SL[DXR]、chTNT-3-SL[DXR]和（chTNT-3/B+SAv-SL[DXR]）的t_1/2分别为21.9h，21.6h和15.0h；AUC_0-72分别为22411μg·h/mL，1440μg·h/mL和1138μg·h/mL；MRT_0-72分别为20.1h，16.9h和13.8h。荷瘤裸鼠组织分布结果表明，预定位脂质体组4h和24h在肿瘤部位的阿霉素浓度最高，4h时显著高于SL组和游离阿霉素组（P＜0.05）；48h空间稳定免疫脂质体在肿瘤部位的浓度最高。荷瘤裸鼠药效试验表明，chTNT-3-SL[DXR]和chTNT-3/B+SAv-SL[DXR]抗皮下移植瘤各有优点，前者的后续药效较好（相对体积抑瘤率59.5％），后者的某个中间时相段药效较好（相对体积抑瘤率61.9％），高于非免疫SL[DOX]（相对体积抑瘤率49.1％）。更多还原

【Abstract】 Liposomes are composed of phospholipid bilayer structures that encapsulate an aqueous interior. Liposome properties and biological behaviors can be controlled by surface modification. Attachments of hydrophilic polymers, such as poly （ethylene glycol）, enable them to have a prolonged circulation time without being opsonized and recognized by mononuclear phagocytic system. Attachments of specific ligands, such as monoclonal antibody or peptides, make liposomes own active targeting effect to specific cells or tissues.Chimeric Tumor Necrosis Therapy -3 monoclonal antibody （chTNT-3） is a novel monoclonal antibody （MAb） which can bind to degenerating cells located in necrotic regions of solid tumors. Since 50-90% of the progeny of tumor cells shortly undergoes degeneration and cell death, chTNT-3 can be utilized to direct at nuclear antigens accessible in necrotic areas of various tumors, while conventional tumor cell MAbs can only target to one kind of tumor cell antigen.Lipid materials for preparing sterically stabilized liposomes, such as methoxypolyethyleneglycol-hydrogenated soy phosphatidyl-ethanol-amine （mPEG-HSPE） and pyridylthiopropionoylamino-PEG-hydrogenated soy phosphatidylethanol-amine （PDP-PEG-HSPE）, were synthesized and determined via TLC, IR, ¹H-NMR, etc.Preparation of active targeting liposomes were carried out via two steps: （1）. The homogenized sterically stabilized liposomes （PDP-SL） with PDP-PEG-HSPE as lipid material were prepared via high pressure extrusion method. The antitumor agent, doxorubicin was then encapsulated using ammonium sulfate gradient method. According to the indexes of encapsulation efficiency and drug load, via central composite design-response surface methodology, an optimized liposome formulation was obtained as follow: HSPC/CHOL/mPEG-HSPE/PDP-PEG-HSPE = 5:4:0.2:0.05 （molar ratio）, drug-lipid ratio=0.18:1 （w/w）. The temperature and time for encapsulation were 65℃C and 20 min. The encapsulation efficiency was about （93.2±2.0）%. （2）. PDP-SLs were reduced by dithiothreitol （DTT） to form thiol groups on the surface of liposomes （HS-SL）. MPB-chTNT-3 were then linked to HS-SLs to form the stabilized immunoliposomes （chTNT-3-SL）. Besides this, SAv-SL could be obtained by linking MPB-streptavidin （MPB-SAv） to HS-SL. chTNT-3 could be derivatized to chTNT-3/B with biotin substitute degree between 3-8. Both chTNT-3/Band SAv-SLs composed of pretargeting preparations.After antibody or SAv modification, the average sizes of chTNT-3-SL and SAv-SL were increased from 102 nm （PDP-SLs） to 123 and 114 nm, respectively. The binding efficiencies of antibody or SAv on liposome surfaces were 69.8% and 82.6%. The surface densities of antibodies or SAv proteins were 108.9 and 51.5μg/μmol PL. The immunoreactivity of chTNT-3 derivatives were determined by ELISA, and the results showed that it was well remained. After being conjugated to the surface of liposomes, the immunoreactivity was kept as well to a certain extent. The preliminary stability test indicated that after 14 days of storage at 4℃, the mean sizes and size distributions of both chTNT-3-SL and SAv-SL changed little, the drug leakage was less than 3%, the relative immunoreactivity of chTNT-3-SL kept unchanged and the physical and chemical properties were stable.To verify the in vitro targeting effect of active targeting liposomes, fixed Raji cell models were established and calcein loaded active targeting liposomes were prepared. The specific binding effects of both sterically stabilized liposomes and pretargeting preparations to fixed Raji cells were investigated by fluorescence assay. The results showed that the binding effects of two kinds of liposomes were both higher than those in control groups （P<0.001）.Followed two-compartment model, the biological half-life of chTNT-3-SL[DXR] and （chTNT-3/B + SAv-SL[DXR]） in rats was longer than that of free doxorubicin but shorter than sterically stabilized liposomes. ChTNT-3-SL[DXR] could target to the necrosis site of tumor and its accumulation in tumor was a gradual process. Pharmacokinetic parameters of rats after iv DXR and DXR preparations were as follow: t_1/2 of SL[DXR], chTNT-3-SL[DXR] and （chTNT-3/B + SAv-SL[DXR]） were 21.9h, 21.6h and 15.0h, respectively; AUC0.72 of them were 2241μg·h/mL, 1440μg·h/mL and 1138μg·/mL, respectively; MRT_0-72 of them were 20.1 h, 16.9h and 13.8h, respectively. In biodistribution investigation of mice bearing H460 tumor, the DXR concentration of pretargeting[DXR] group was the highest in all groups at 4h and 24h and was significantly higher than SL[DXR] and free DXR groups at 4h （P<0.05）. And at 48h the DXR concentration of chTNT-3-SL[DXR] group was highest in all groups. The advantages of antitumor efficacy of chTNT-3-SL[DXR] and chTNT-3/B+Strepavidin-SL[DXR] pretargeting preparations to subcutaneous transplantation tumor varied. ChTNT-3-SL[DXR] had a better antitumor activity with a final tumor inhibitory rate of 59.5%, while the pretargeting preparations had thehighest antitumor efficacy with tumor inhibitory rate of 61.9% during some intervals, which was better than that of SL[DXR]’s （49.1%）.更多还原