【作者】 李博；

【导师】 金一；

【作者基本信息】 浙江大学 ， 药剂学， 2012， 博士

【摘要】 肿瘤血管新生在肿瘤生长和转移过程中起着关键性作用。近年来,新生血管抑制剂与化疗药物被联合用于肿瘤的治疗,并取得了良好的临床治疗效果。但这种药物的联合使用在临床也产生了新的问题,比如：肿瘤血液供应的切断,会导致化疗药物无法达到肿瘤组织,从而影响药效的持续性发挥；其次,肿瘤部位营养和氧气供应的切断,会使肿瘤细胞表达更多的缺氧耐受因子,使肿瘤细胞的侵袭力增加,从而容易诱发肿瘤转移,增加肿瘤治疗的难度。为了解决肿瘤联合给药出现的问题,本文制备了具有核-壳结构的核壳型脂质纳米粒(LNP)程控式给药系统,在纳米粒核中负载阿霉素(DOX),脂质体外壳负载双氢青蒿素(DHA)以实现DHA和DOX的先后释放,抑制肿瘤血管新生的同时,实现在肿瘤组织持续化疗的目的。此外在肿瘤新生血管内皮细胞上,具有高表达的αvβ3受体,c(RGD)fk肽可以与之特异性结合。利用这一性质,我们以c(RGD)fk肽为肿瘤特异性配体,制备了c(RGD)fk-聚乙二醇-胆固醇(Chol-PEG-c(RGD)fk)接枝共聚物,并将其修饰到双载药的核壳型脂质纳米粒(D-LNP)的表面,以提高肿瘤部位靶向性,减少毒副作用。所制备制剂不但实现了药物的梯度释放,而且可以通过肿瘤血管的靶向性,直接将药物运输到肿瘤组织,提高制剂的作用效率。相关研究工作的主要内容及结果如下：首先,制备给药系统的“核”-DOX-PLGA纳米粒。利用两步反应制备DOX-PLGA接枝共聚物,并用核磁共振、GPC等方法对其结构进行表征。以双十二烷基二甲基溴化铵为乳化剂,采用乳化分散挥发法制备DOX-PLGA纳米粒,考察了不同工艺对纳米粒粒径、载药量和包封率的影响。结果表明,纳米粒粒径受乳化剂浓度、有机溶剂种类的影响,粒径分布在100～200 nm,zeta电位为57.0～60.4 mV。载药量和包封率不受粒径大小的影响。透射电镜结果表明,PLGA纳米粒呈球形,大小均一；扫描电镜结果表明PLGA纳米粒表面形态光滑,为刚性结构。X射线衍射的结果表明,阿霉素以无定形形式存在于纳米粒中。体外释放结果表明,阿霉素PLGA纳米粒在偏酸性环境中的释放较快。稳定性研究表明,纳米粒溶液相对稳定,增加溶液的离子强度,有利于纳米粒的收集。然后,采用薄膜分散法制备具有核-壳型结构的双载药LNP,并考察纳米粒浓度,药脂比等因素对DHA载药量和包封率的影响。结果表明,随着纳米粒比表面积和磷脂浓度的增加,DHA的包封率和载药量均有所提高,最大载药量为3.58%。LNP的平均粒径分布在100～300 nm, zeta电位在-8.5～-6.3 mV之间。之后,对LNP进行功能化修饰。首先合成Chol-PEG-c(RGD)fk接枝共聚物,通过红外光谱、核磁共振、GPC对其进行表征；然后将Chol-PEG-c(RGD)fk偶联到核壳型脂质纳米粒表面。透射电镜观察LNP和c(RGD)fk-LNP的形态,结果表明,核壳型脂质纳米粒呈球形,分布均匀,脂质外壳厚度约为20 nm。扫描电镜的结构表明核壳型脂质纳米粒表面出现了退行性变化,表现为脂质体的囊泡结构特征。X射线衍射结果表明,两种药物均以无定形的形式存在于纳米粒中。激光共聚焦显微镜结果表明,阿霉素标记的PLGA内核可以与NBD-PE标记的磷脂外壳完全的重合。小角度X射线散射衍射结果表明,LNP存在着核壳型结构,并且Chol-PEG-c(RGD)fk的表面修饰使磷脂外层的结构发生改变。X射线光电子能谱的结果表明,阿霉素PLGA纳米粒被完全包裹在磷脂层内。体外释放结果表明,双氢青蒿素与阿霉素在核壳型脂质纳米粒制剂中为程序性梯度释放,这有利于核壳型脂质纳米粒协同发挥新生血管抑制剂和化疗制剂的疗效。核壳型脂质纳米粒的稳定性研究表明,LNP和c(RGD)fk-LNP的粒径在溶液中有增加趋势,应冷冻干燥保存。采用MTT法评价了空白PLGA-NP,LNP和c(RGD)fk-LNP的细胞毒性,结果表明磷脂层的包裹可以改善阳离子纳米粒的生物相容性,所制备的LNP基本无细胞毒性。同时考察了游离阿霉素,游离双氢青蒿素以及两者配伍使用时对肿瘤细胞的细胞毒性作用,结果表明,游离DOX与DHA均有细胞毒性,DHA对细胞无选择性,但DOX对耐药株毒性较弱。DHA与DOX可显著提高DOX的抗肿瘤效率,两者协同作用可在一定程度上逆转MCF-7/ADR细胞的耐药性。结合激光共聚焦显微镜和流式细胞术结果,可以看出,通过核壳型核壳型脂质纳米粒的负载,DOX可靶向蓄积在肿瘤细胞的细胞核。这种胞内的分布可能与DMAB分子可以引起内吞体膜的破裂有关。载体的细胞核靶向有利于进一步的增加药物的细胞毒性,提高对耐药细胞的逆转效率。采用荷S180瘤ICR小鼠模型,建立HPLC检测小鼠血浆和组织样品的阿霉素含量测定方法,考察核壳型脂质纳米粒在荷瘤小鼠体内的组织分布。结果表明,LNP组和c(RGD)flk-LNP组的AUC0.∞是阿霉素组的2.43倍和2.71倍,c(RGD)fk介导的LNP可以降低消除速率,延长平均滞留时间,增加载体在肿瘤组织的分布,提高靶向性,降低在心脏和肾脏的药物分布及毒性。此外,核壳型脂质纳米粒与游离的阿霉素相比,在肝脏,脾脏等网状内皮系统的聚集增加。最后,构建小鼠的S180移植瘤和B16肺转移瘤模型,进行核壳型脂质纳米粒抗肿瘤的药效学研究。结果表明,c(RGD)fk介导的核壳型脂质纳米粒组对S180的抑瘤率可达79.5%,远高于阿霉素与双氢青蒿素的混合物；对B16黑色素转移瘤也有明显抑制效果。对小鼠肿瘤的免疫组化研究表明,DHA可以下调COX-2的表达,进而抑制肿瘤的血管生长,使MVD值减小。但血管的生长抑制同样会引起缺氧耐受因子(HIF)的高表达。c(RGD)fk介导的核壳型脂质纳米粒组可以通过药物的程序性释放改善由血管新生抑制治疗引起的HIF高表达,体现了良好的治疗效果。此外心脏毒性研究发现,核壳型脂质纳米粒制剂可以显著降低阿霉素的心脏毒性。更多还原

【Abstract】 Angiogenesis plays an important role in the tumor growth and metastasis. Re cently, excellent clinical results have been achieved by co-administration of angio genesis inhibitor and chemotherapy drug. However, these combined treatments als o produce new clinical problems. Such as, the cut-off of tumor blood supply wil 1 lead to low concentration of chemotherapy drugs in tumor tissue to decrease th e effiecacy of chemotherapy; the lack of nutrients and oxygen will also increase the expression of oxygen tolerace factor, which may cause tumor metastasis. To solve the problems from the combined administration, a novel drug delivery syst em called lipid/polymer particle assemblies was prepared here. Lipid/particle asse mblies named LipoParticles (LNPs) consisting of dimethyldidodecylammonium bro mide modified ploy (lactic-co-glycolic acid) nanoparticles core surrounded by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine shell were specially designed for the com binatorial treatment. The dual drugs loaded LNPs can cause sequential release of DHA and DOX, killing endothelial cells and tumor cells respectively and increa sing cancer treatment effect. To improve the LNPs targeting to tumor, we synthe sized the Chol-PEG-c(RGD)fk conjugates to modify the drug delivery system.The DOX-PLGA conjugates were prepared and characterized by 1H NMR and GPC. Then the conjugates were used to prepare DOX nanoparticles using the emulsification-diffusion method. The mean diameters of these nanoparticles were in a range of 100-200 nm with a zeta potential of 57.0～60.4 mV. Transmission electron microscopy (TEM) showed relatively uniform sizes of DOX-NP and scanning electron microscopy (SEM) showed that the surface structure of the PLGANPs modified by DMAB was fairly rigid, which did not show the decomposition. The in vitro release behavior of DOX-NP was accelerated by increasing acidic surrounding and presented a biphasic release pattern. Stablilty experiments revealed that the nanoparticles solution was relatively stable. Increases ionic strength was conducive to the collection of nanopartlces.Dual drug delivery LNPs were prepared by film ultrasonic dispersion method. The effects of different concentrations of nanoparticles, ratio of lipid to drug on drug loading content and encapsulation efficiency were determined. The results showed that the max DHA loading content of LNPs with mean diameter of 100～300nm, was 3.58%, zeta potential of -8.5～6.3 mV. The Chol-PEG-c(RGD)fk conjugates were prepared and characterized by FT-IR,1H NMR and GPC. Then, these conjugates were used to modify LNPs. Transmission electron microscopy (TEM) showed the relatively uniform sizes of LNPs, which were ringed by a layer of petal-like vesicle with bilayer thickness of about 20 nm. Scanning electron microscopy (SEM) showed that the LNPs’surface presented the degradation and crack by electron beam bombardment effect. These results well demonstrated that the nanoparticles were covered by a layer of DPPC. Confocal microscopy showed that doxorubicin labeled PLGA core and NBD-PE labled phospholipid shell perfect coincidence. Small-angle X-ray scattering diffraction results also showed LNPs’core-shell structure, and the surface structure was changed by the modification of c(RGD)fk-PEG-Cholto phospholipid layer. X-ray photoelectron spectroscopy for surface elemental analysisi results showed that the PLGA naoparticles were completely wrapped into the phospholipid layer. In vitro release studies showed that the procedural release behavior of DHA and DOX in the LNPs. The stability studis revealed that LNP should be freeze-dried.Cytotoxicity of blank LNP and c(RGD)fk-LNP against tumor cells wad evaluated by MTT assay. The results showed that the DPPC shell could improve biocompatibility, and the blank LNP and c(RGD)fk-LNP did not have significant cytotoxicity. Also, the cytotoxicity of DOX, DHA and the mixture of DOX and DHA to tumor cells was investigated. The DHA could improve anti-tumor efficiency of DOX. MTT results of MCF-7/ADR study results showed that the cooreinated use of DHA could reverse cell resistant. Based on laser confocal microscopy and flow cytometry results, DOX loaded by core-shell nanoparticles could accumulate in the nucleus of tumor cells, which could further enhance the cytotoxicity of agents and increase the revearsal efficiency in MCF-7/ADR cells.High performance liquid chromatography (HPLC) was developed to determine the content of DOX in plasma and tissues of S180 tumor-bearing ICR mice. In vivo tissue distrubuton was investigated. The results showed that c(RGD)fk-mediated tumor vascular targeting LNPs could extend the mean residence time and reduce the emlimination rate. Thus, the distribution of DOX in tumor tissue could be significantly increased, AUC0-∞of LNP and c(RGD)fk-LNP group were 2.43 and 2.71 times higher than DOX group. DOX package in LNPs decreased the distributionin heart and kidney to reduce cardiac toxicity and kidney toxicity, which also explained the long-term retention in the body. In addition, compared to free DOX, LNPs showed reticulo endothelial system accumulation.Mouse S180 xenograft model and B16 lung metastases model were used to establish anti-tumor efficiacy. The results showed that the inhibitory rate of S180 tumor could reach 79.5%, which was much higher than DOX and the mixture of DHA-LP and DOX-NP. B16 melanoma metastases had also been significantly controlled. Immunohistochemical studies in S180 tumor had shown that DHA could reduce COX-2 expression to inhibit tumor angiogenesis and MVD. However, this would also lead to high expression of HIF. c(RGD)fk-LNPs improved this combination treatment induced expression of HIF by gradient drug release of DHA and DOX. In addition, cardiac toxicity studies in B16 melanoma metastatic lung mice found that LNPs could significantly reduce the cardiotoxicity of DOX.更多还原