【作者】 刘利萍；

【导师】 蔡绍皙； 吴泽志；

【作者基本信息】 重庆大学 ， 生物医学工程， 2003， 博士

【摘要】 磁性导向给药系统是由高分子材料负载抗癌药物和磁响应性物质构成，在足够强的体外磁场引导下通过血管等选择性地达到并定位于肿瘤靶区释放药物，使药物在肿瘤组织的细胞或亚细胞水平发挥药效作用，而对正常组织无太大影响。它为抗癌药物的靶向性提供了一种新途径。本论文研究目的：以生物相容性好的新型可降解天然高分子材料-丝素/壳聚糖（FB/CS）复合物为载体，氟尿嘧啶（5-Fu）为模型药物，探索5-Fu靶向缓释磁微球的制备工艺、工艺参数与微球性能的相关关系。在此基础上研究氟尿嘧啶磁性丝素/壳聚糖复合微球（5-Fu-MFCMs）在模拟人体介质中的药物释放规律、磁微球定位的生物力学机制和对不同肿瘤细胞株的杀伤动力学行为，为其应用提供实验与理论指导。现已获得如下进展：（1）以PEG-4000作分散剂，用化学共沉淀-超声法制备水基磁纳米粒子。该磁粒子近似球形，粒径分布范围为25±10nm，饱和磁化强度为36.2×103A/m，属于超顺磁性物质。该磁纳米粒子具有稳定性高、磁响应性强、与亲水性高分子材料有较强亲和力的特点。（2）改性丝素合金膜中的FB与CS分子间存在着强烈的氢键相互作用及良好的相容性，其对应等电点的pH值是5.35，而纯FB膜等电点的pH值为4.5。当CS含量为40～60%时，改性丝素合金膜具有最大的抗张强度（71.4～72.7MPa）和拉伸率（3.44～3.82%）。改性丝素合金膜对5-Fu的渗透量与合金膜中CS的含量和渗透时间呈正相关，渗透系数随pH的增大（5→9）先是逐渐减小然后略有增大，在pH=7时最小，该值与人体生理环境相近，可作为pH智能响应膜。（3）建立了5-Fu-MFCMs磁微球的消解和5-Fu含量的一阶导数光谱测定方法。分别用酸碱消解微球以释放所包裹的5-Fu，在酸性介质中，最大波谷D对应的波长为280nm，；在碱性介质中，最大波谷D对应的波长为299nm，。二者的线性浓度范围均是3.36～16.8μg/ml，平均回收率为101.83±0.68%，RSD为0.67%。该方法不需分离杂质，具有简单方便、重现性好的特点。（4）采用改良的W/O乳化交联技术制备5-Fu-MFCMs微球。通过正交设计、引入“归一化值”OD并利用统计学分析方法，得到了工艺参数与微球各性能指标（如粒径、载药量、包封率、磁响应性、缓释性及“归一化值”OD）之间的多元线性回归或二项式拟合方程（R＞0.9），其中对OD有显著性影响的因素（P＜0.05）按大小顺序依次是：固化时间X7＞搅拌速率X2＞载体浓度X7＞原料配比X5＞乳化剂浓度X4＞固化<WP=5>剂浓度X5。且各工艺因素之间有一定的交互作用，大小顺序是：X5X7＞X5X6＞X1X6＞X1X4＞X2X7＞X2X6除乳化剂浓度与微球的OD呈负相关外，其它因素均与OD呈正相关。即适当减小乳化剂浓度，增大载体浓度、乳化速率、固化时间、原料配比和固化剂用量可得到综合指数OD较高的含药磁微球。按优化工艺参数制备微球的实验值与按回归方程的预测值有较好的吻合性。所得5-Fu-MFCMs的载药量与包封率分别为12.11%和60.55%、磁响应性为8s（指在0.4T磁场作用下平行移动10cm所需时间）、在体外模拟介质中释药50%的时间t50为11.72hr、综合指数OD为0.6573。电镜下观察微球表面光滑、圆整、具有良好的分散性，平均粒径为4.36±1.92，分布在1～7的微球占总量的91.04%。经红外、差热和X衍射检测，5-Fu与丝素蛋白/壳聚糖合金物载体之间以很强的分子间相互作用力形成了共沉淀物。（5）以该工艺及FB/CS复合物为负载材料制备的5-Fu-MFCMs微球，药物在0.5hr内存在突释现象，随后呈现出缓慢释放的特点。FB/CS复合高分子载体属于可溶胀聚合物系统，对5-Fu的释放遵循Fick扩散机理，具有骨架型微球的释药特征，其体外释药动力学模型符合Higuchi方程，拟合相关系数R均在0.9以上。（6）模拟血流环境，建立了磁微球在磁场作用下穿过流体时定位的力学模型，得出影响磁微球定位因素的相互关系。即对粒径和磁性一定的磁微球，产生定位的磁场条件为：＞根据实验结果并结合理论预测，对平均粒径为4.3含磁粒子20%左右的磁性微球，当外磁场强度H≥0.4T、介质流速≤1cm/s、靶区离磁场距离≤3cm时，磁微球在靶区被截留率可达75%以上。（7）用MTT比色法测定不同时间剂量的5-Fu-MFCMs微球对人肝癌细胞株SMMC7721与肺腺癌细胞株A549的杀伤活性。结果表明5-Fu-MFCMs磁微球对肿瘤的药理作用与5-Fu相似，而未包裹药物的空白磁微球无抗肿瘤活性。5-Fu-MFCMs对肿瘤杀伤活性与作用时间和释药量呈正相关关系。根据半数抑制剂量IC50，5-Fu-MFCMs对肺腺癌A549较人肝癌SMMC7721更为敏感更多还原

【Abstract】 Magnetic targeted drugs delivery system consists of polymer carrier, drug and magnetic nanoparaticles. It could be selectively localized to the target-site of tumor by external magnetic field and then the drug would be delivered to cell and sub-cell of tumor tissues. Magnetic microspheres agent loaded antitumor drug is suggested as an efficient method of obtaining high local drug concentration and may reduce many undesirable toxic side effects from unrestricted systemic circulation.The aim of the thesis is that biodegradable silk fibroin modified by blending with chitosan as vehicle are prepared target microspheres formulation with a high loading efficiency for the cytotoxic drug 5-fluorouracil (5-Fu) and magnetic nanoparticles. The interrelationships are studied between phy-chemical preparation parameters and technical indexes of microspheres. It is studied that the kinetic release model of 5-Fu from fibroin/chitosan microspheres, influential factors to microspheres localized sites by external magnetic field and its antitumor activities to tumors, respectively. Some achieved progresses have been obtained as follows: (1)With a chemical coprecipitation and ultrasonic technology, ferric/ferrous ion salt dispersed in polyethylene glycol-4000(a dispersant agent), and then ammonia water added to pH 10～11 of solutions, magnetic nanoparticles were obtained. Measuring results of scanning tunnel microscope showed that the particles were round with a size range of (25±10nm). Its saturation magnetization was 36.2×103A/s and it belongs to superparamagnetism of ferrofluid. The magnetic nanoparticles exhibited excellent stability, strong magnetic response and well affinity with hydrophilic polymer.(2) The Modified fibroin membranes were studied some characters to be found such as there was a strong hydrogen bond and good compatibility between fibroin and chitosan molecules, and its isoelectric point at pH=5.35, but that of fibroin film was around pH 4.5. It was also found that its mechanical properties were much higher than those of fibroin or chitosan film. Its tensile strength and breaking elongation were greatly enhanced with increasing the chitosan content and showed maximum values of 71.4～72.7MPa and 2.96～3.82% respectively at containing chitosan 40～60%. Its coefficient of permeability to 5-fluorouracil was decreased initially and then increased slowly from pH 5 to 9 of solutions with the minimum located at pH=7.(3) The first order derivative spectrophotometry method for the determination of<WP=7>5-Fu content in magnetic fibroin/chitosan blended microspheres was established. Blended magnetic microspheres were digested with HAc and NaOH respectively to release entrapped 5-Fu. The absorption value of amplitude at the wavelength of 280nm in 10%HAc and at 299nm in 0.1 mol/l NaOH was used. The interference of fibroin and chitosan could be ignored. Its regression equations were ，(in HAc) and (in NaOH) respectively. The average recovery was 101.83±0.68% and RSD was 0.67%. This method is simple, rapid and well reproducible.(4) A novel W/O emulsion/polymer cross-linking technique was used to prepare magnetic fibroin/chitosan microspheres containing 5-Fu (5-Fu-MFCMs). An orthogonal experimental design and statistical analysis methods were employed to simultaneously optimize and evaluate a variety of formulation factors on a number of response variables. OD value was the overall desirability of multiple response variables, such as mean diameter and dispersity efficiency, drug loading and loading efficiency, magnetic response and 50% release time etc. Second-order polynomial and linear equations were fitted to the data, and the regression equations and their correlation coefficients R＞0.9 were obtained. The statistical optimization procedures indicated the sequence of the factors of significantly influence to OD（P＜0.05）was below:Crosslinking time＞Stirring rate＞FB/CS concentration＞5-Fu /magnet /FB-CS ratio＞Emulsifier concentration＞Cross-linker concentration.Most of these factors above were posit更多还原