【作者】 游本刚；

【导师】 张学农；

【作者基本信息】 苏州大学 ， 放射医学， 2013， 博士

【摘要】 本文以肝肿瘤细胞上的ASGPR和CD147分子为靶标，采用N-乳糖酰壳聚糖（GC）和CD147单抗分别实现受体介导与抗体介导，以α-常春藤皂苷为模型药物，将其制备成“受体+抗体”的双重复合机制介导的肝肿瘤主动靶向纳米给药系统。采用乳化溶剂扩散法制备了α-Hed-GC-NP，考察并优化了纳米粒的处方和制备工艺，确定影响纳米粒质量的关键要素为GC用量：50mg，磷脂用量：80mg，有机相体积：4mL。所制得的α-Hed-GC-NP外观圆整、分布均匀，平均粒径（88.70±1.22）nm，包封率（78.53±4.39）%，P.I.值0.105±0.011；药物在载体材料中分散均匀，释药行为具有一定的pH敏感性和缓释特性。建立了CD147单抗的含量和活性测定方法，采用偶联法、溶解法、注入法、吸附法及修饰法等5种方法制备了α-Hed-GC-CD147-NP，分别进行粒径测定、FT-IR表征和纳米粒中单抗结合活性测定。结果表明，CD147均被α-Hed-GC-NP有效包载，采用修饰法制备的α-Hed-GC-CD147-NP，平均粒径为（139.53±4.17）nm，CD147单抗的修饰量为1.25μg/mgNP，单抗的结合活性保留率为52.29%。对α-Hed-GC-CD147-NP的细胞毒作用、诱导细胞凋亡、细胞摄取率及摄取过程进行考察，并与其它3种纳米粒进行比较。结果表明，α-Hed及其含药纳米粒对SMMC-7721和HepG2两种细胞均有显著的抑制作用，α-Hed-GC-CD147-NP对两种细胞的IC50最低，分别为（23.51±1.22）、（23.30±0.22）μg/mL；纳米粒能诱导细胞凋亡，并呈现剂量依赖性，α-Hed-GC-CD147-NP低、中、高剂量（50、100、200μg/mL）诱导HepG2细胞的凋亡率分别为7.10%、11.87%、25.78%，高于其他纳米粒；HepG2细胞对α-Hed-GC-CD147-NP的细胞摄取率最高，2h和24h分别达（86.73±11.29）%和（94.61±4.42）%；随着时间的延长，纳米粒大部分被摄入到包浆，并能进入细胞核，α-Hed-GC-CD147-NP与HepG2细胞的亲和力更强，具有良好的肝肿瘤细胞靶向性。研究了α-Hed-GC-CD147-NP的急性毒性，对荷HepG2移植瘤裸鼠的抗肿瘤活性及体内靶向性。结果表明，各纳米粒小鼠尾静脉注射LD50均在12mg·kg-1以上，高于原料药；各剂量组对荷HepG2细胞裸鼠肿瘤的抑制率IRw均在70%以上，远高于原料药中剂量组；具有双重复合介导机制的α-Hed-GC-CD147-NP抑瘤率最高，低、中、高3个剂量组的抑瘤率分别为84.96%、88.25%、92.46%，显示了更好的肝肿瘤靶向性。荷瘤裸鼠近红外荧光成像实验结果表明，经受体或抗体介导的纳米粒，肿瘤和肝脏靶向性更强。建立了UPLC-MS法测定体内血浆及组织样品中α-Hed的分析方法，考察α-Hed-GC-CD147-NP在大鼠体内的药物动力学特征，及在荷瘤裸鼠体内组织分布情况。结果表明，药物在大鼠体内呈双室模型，α-Hed-GC-NP、α-Hed-CS-CD147-NP及α-Hed-GC-CD147-NP的t1/2(α)分别为0.12、0.10、0.05h，t1/2(β)分别为0.97、2.30、1.93h，与原料药相比，各纳米粒体内分布、消除减慢。以α-Hed的AUC为参照，上述3种纳米粒的生物利用度显著提高，分别为120%、300%及280%。抗体或受体介导的纳米粒，在肝脏和肿瘤组织内的药物量远远高于其他组织，具有极显著性差异，显示了良好的肝脏和肿瘤靶向性。双重复合机制介导纳米粒α-Hed-GC-CD147-NP在荷瘤裸鼠的肝脏和肿瘤内的分布量最高，优于单一抗体或受体介导纳米粒。更多还原

【Abstract】 Based on the specific recognition ability of galactosylated chitosan (GC) toasialoglycoprotein receptor (ASGPR), identified as a target antigen on hepatoma cells,and of CD147mAb to CD147, over-erpressed on the surface of malignant cells,receptor-mediated active targeting and antibody-mediated active targeting werecombined to achieve nano drug delivery system of α-Hederin nanoparticles associatedwith GC and CD147targeted to hepatocellular carcinoma.Formulation and technology of α-Hed-GC-NPs prepared by emulsion solventdiffusion method were investigted and optimized, and key factors were identified to bedosage of GC (50mg), dosage of lecithin (80mg), and organic phase volume (4mL).α-Hed-GC-NPs optimized were presented a spherical morphology and a unimodal andrelatively narrow particle size distribution, the mean particle diameter was88.70±1.22nm, and its polydispersity index was0.105±0.011, and the average entrapmentefficiency was78.53%±4.39%. Characterization of α-Hed-GC-NPs indicated thatα-Hed were well dispersed in GC carriers, and in vitro release profile showed that drugrelease from nanoparticles was sustained and slightly dependent on pH.α-Hed-GC-CD147-NPs were prepared and compared by coupling method,dissolution method, injection method, adsorption method and modification method,respectively. Particle size determination showed that the mean particle diameters ofα-Hed-GC-CD147-NPs were significantly increased compared with α-Hed-GC-NPs,and the mean particle diameters of nanoparticles prepared by modification method was139.53±4.17nm. Characterization of α-Hed-GC-CD147-NPs by Fourier transforminfrared (FT-IR) spectrophotometer revealed the formation of Intra-and inter molecularhydrogen bonds, indicating the well dispersion of drug in nanoparticles. The conjugatedamount of CD147mAb to α-Hed-GC-CD147-NPs prepared by modification method was1.25μg/mgNP, and the conservation rate of binding activity of mAb was52.29%,prior to nanoparticles prepared by other methods.Cytoxity of α-Hed-GC-CD147-NPs was investigated and compared with other3nanoparticles, α-Hed and its drug-containing nanoparticles can significantly inhibitedthe growth of HepG2and SMMC-7721cells, the inhibitory ability of α-Hed-GC-CD147-NPs was highest, IC50to SMMC-7721and HepG2was23.51±1.22μg/mL and23.30±0.22μg/mL, respectively. Nanoparticles could induce apoptosis of HepG2celland apoptosis rate depended on the dose of α-Hed. The apoptosis rats induced byα-Hed-GC-CD147-NPs at a dose of50,100, and200μg were7.10%,11.87%and25.78%, respectively, higher than that of other nanoparticles. Cellular uptake rates at2hand24h of HepG2cells for α-Hed-GC-CD147-NPs were86.73±11.29%and94.61±4.42%, respectively. With the extension of time, most of nanoparticles wereintaked into slurry, and had access to the cell nucleus, α-Hed-GC-CD147-NPs had astronger affinity to HepG2cell and better ability targeting to liver tumor cells.Acute toxicity in mice by tail vein injection of α-Hed and4nanoparticles wastested and the LD50were above12mg/kg, more than α-Hed. Inhibition rates of tumorweight in HepG2-bearing nude mice of4nanoparticles for3dose groups (0.5,1.0,2.0mg/kg) were all above70%, more than α-Hed (1.0mg/kg). Inhibition rates ofα-Hed-GC-CD147-NPs at doses of0.5,1.0,2.0mg/kg were84.96%,88.25%and92.46,individualy, showed the better liver tumor targeting ability, which was also confirmedby infrared fluorescence imaging.UPLC-MS method for determination of α-Hed in plasma of rats and tissuehomogenate samples of nude mice, and pharmacokinetics in rats and distribution inHepG2-bearing nude mice of α-Hed-GC-CD147-NPs were investigted. α-Hed in ratsshowed a two-compartment model, for α-Hed-GC-NPs, α-Hed-CS-CD147-NPs andα-Hed-GC-CD147-NPs, t1/2(α) were0.12,0.10,0.05h, respectively, t1/2(β) were0.97,2.30,1.93h, respectively. Compared with α-Hed, drug distribution and elimination ofnanoparticles was slowed. Using the AUC of α-Hed as the reference, bioavailability of3nanoparticles above were significantly increased20%,200%and180%respectively.For nanoparticles mediated by antibody or receptor, drug amounts in the liver and tumor tissue is much higher than other organizations, which showed a favorable targetingability to liver or tumor. α-Hed-GC-CD147-NPs, mediated by dual mechanismcombined the receptor-mediated and antibody-mediated active targeting mechanism,drug distribution in liver and tumor of HepG2-bearing nude mice was the highest, andbetter than nanoparticles mediated by single receptor or antibody.更多还原