【作者】 张良珂；

【导师】 侯世祥；

【作者基本信息】 四川大学 ， 药剂学， 2004， 博士

【摘要】 卵巢癌是女性常见的肿瘤之一,极大地危害了女性的健康,目前治疗手段不甚理想,因此设计一种能将药物靶向于卵巢癌细胞的给药系统更具有实际意义。 靶向给药系统(Targeting drug delivery system)是药剂学研究的热门领域。经过长期研究,人们对靶向给药系统的靶向机理、特性、体内分布和代谢规律都有了较清楚的认识。但是,有关抗癌药物靶向给药系统的研究,大多以靶向至肿瘤所在组织器官为目的,如何使更多的药物达到肿瘤部位后进入肿瘤细胞,减少对肿瘤所在器官的毒性,真正达到肿瘤细胞靶向给药的目的,是近几年靶向给药系统的研究重点。 受体和其配体的结合具有特异性、选择性、饱和性、亲合力强和生物效应明显等特点。利用配体为药物或放射性核素的载体,通过受体介导作用,增加药物在病灶局部的浓度、提高疗效,降低毒副作用,达到靶向治疗目的,是目前研究最活跃的前沿领域之一。 随着对细胞膜表面叶酸受体(一种被磷脂酰肌醇锚在细胞膜上的叶酸结合蛋白,可通过特异性磷脂酶C或D将其切除)认识的逐步深入,发现在多种肿瘤细胞(如卵巢癌、结肠直肠癌、乳腺癌、肺癌和肾细胞癌等)膜表面上的叶酸受体活性和数量显著高于一般正常细胞,为叶酸介导药物靶向肿瘤细胞的研究奠定了基础。 白蛋白具有可生物降解及无毒无免疫原性的特点,是一种常用的靶向载体材料。以有机溶剂对白蛋白去溶剂化再进行交联固化是制备白蛋白纳米粒的一种常用的方法。白蛋白分子中赖氨酸数目众多(约占残基更多还原

【Abstract】 Ovarian cancer is a common cancer of organa genitalia feminine, which do harm to the health of women greatly. Now there are no good therapy, so it is practical to design a targeting delivery system to delivery chemical drugs to the ovarian cancer cells.Targeted drug delivery system (TDDS) is a hot issue in Pharmaceutics After long time pursuing, people become to realize the targeted mechanism, property, biodistribution and pharmacokinetics of TDDS. However, most of the anti-cancer drug delivery systems are aimed at being targeted to the organ where tumor is localized. How to target the therapeutics directly to tumor without distribution to surrounding tissues, reduce their side effect, improve their therapeutic efficacy, is one of the most important direction for TDDS.Using receptors as markers may be an advantageous strategy for drug delivery. Receptor-mediated uptake can achieve the specific transport of the drug to the receptor-bearing target cells. The binding between receptor and ligand is specific, selective, saturated, high affinitive, strongly effective. How to increase the concentration of drugs, enhance the curative effect and reduce side effect through receptor-mediated targeting were researched a lot. Many receptors, such as receptors of transferring, low-density lipoprotein, and asialoglycoprotein, have been used to deliver drugs to specific types of cells or tissues.Folate receptor (FR), also known as the high affinity membrane folate-binding protein, is a glycosylphosphatidylinositol (GPI)-linkedmembrane glycoprotein with an apparent molecular weight of 38^0 kDa. The receptor for the vitamin, folic acid, is overexpressed on a number of human tumors, including cancers of the ovary, colon, breast, uterus, lung and kidney. Conjugates of folic acid can bind to and enter receptor-expressing cancer cells via folate receptor-mediated endocytosis. Theoretic foundation of folate receptor-meditated targeting was established.As carrier material, albumin may be very promising because of their biodegradability, lack of toxicity and antigenicity, stability, shelf life, controllable drug-release properties. There are a large number of amino groups in the albumin molecules and on the surface of albumin nanoparticle(surface reactive amino groups) which are the efficient conjugation positionThe bovine serum albumin nanoparticles (BSANP) were prepared by a coacervation method and chemical cross-linking with glutaraldehyde. Single factor design was used to optimize the formula and technology for preparing BSANP and the influence of volume of glutaraldehyde, cross-linking time, concentration of BSA, volume of ethanol, pH were investigated. BSANP were then conjugated to the activated folic acid via surface active amino groups of the BSANP, to improve their intracellular uptake and ability to target specific cells. Then the folate-conjugated BSANPs (BSANP-folate) were purified with Sephadex G-50 column and completely separated from unreacted folic acid. After chymotryptic hydrolysis, the extent of folate conjugation on the BSANP was determined by quantitative ultraviolet(UV) spectrophotometric analysis. The spectrum of trypsin digest of folate-conjugate BSANP is basically identical with that of folate. So folate is conjugated successfully on the surface of BSANP. The influence of reaction time, pH and concentration of N-hydroxysuccinimide ester on the conjugation of folate were investigated by the Single factor design.Cell uptake studies were carried out in SKOV3 cells (human ovariancancer cell line) expressing folate receptors. BSANP-folate labeled with fluorecein isothiocyanate (FITC) and BSANPs were cultured with SK0V3 cells respectively. BSANP-folate were taken up by SKOV3 cells via a saturable mechanism and the BSANP-folate uptake increased as the time went on until no further increase by the 4-h time point. Furthermore, the binding of BSANP-folate to SKOV3 cells could be competitively inhibited by excess free folate. All of those results demonstrate that the interaction was mediated by the cell surface folate receptor.Mitoxantrone was chosen as model drug and folate-conjugated mitoxantrone albumin nanoparticles (MTO-BSANP-folate) were prepared. The drug loading is 9.66% and the embedding ratio is 96.55%. The cytotoxicites of MTO-BSANP-folate, MTO-BSANP, MTO-soln was determined by 3HTdR incorporation assay and the apoptosis was determined by Flow Cytometer. The results show that the cytotoxicity and apoptosis of MTO-soln was stronger than MTO-BSANP due to the molecular structure of MTO, but weaker than MTO-BSANP-folate because of the folate receptor-mediated endocytosis.The SKOV3 tumor model was established on Blab/c-nu mice and the distribution of MTO-BSANP-folate, MTO-BSANP, MTO-soln was investigated by high performance liquid chromatography. Compared with MTO-BSANP and MTO-soln, the targetablity of MTO-BSANP-folate on tumor was elevated. In the experiment of effect of medicine, the rate of tumor inhibition of MTO-BSANP-folate (84.53%) was higher than those of MTO-BSANP and MTO-soln. It was suggested that the MTO-BSANP-folate has ability for active targeting to tumor in vivo to a certain extent.Folate-conjugated albumin nanoparticles are novel tumor intracellular targeting drug delivery system mediated by folate receptor. They were prepared first time in this research. The methods of verification and determination for surface-conjugation were established. Further study on invitro tumor cell uptake and distribution in rumor-bearing mice were carried out. All these studies above-mentioned were not yet reported and this research is quite innovative. A good theoretical and practical foundation was eatablished for exploration of tumor intracellular targeting delivery system on pathway, law, characteristics and mechanism through this research, which not only offered reference and experience for folate-receptor mediated drug delivery system but also brought new ideas to design and develop a new type receptor-mediated drug delivery system.更多还原