【作者】 吴承格；

【导师】 孙康宁； 赵冬梅；

【作者基本信息】 山东大学 ， 材料加工工程， 2014， 博士

【摘要】 膀胱肿瘤(tumor of bladder)是泌尿系统中最常见的肿瘤,绝大多数来自上皮组织,其中90%以上为移行上皮肿瘤。在男性中位居第四位,女性中居第十位。肿瘤的扩散主要向膀胱壁内浸润,直至累及膀胱外组织及邻近器官。为防止肿瘤复发,术后可采用膀胱内药物灌注治疗。常用药物有丝裂霉素、阿霉素、羟基喜树碱及卡介苗(bacillus Calmette-Guerin即BCG)等,目前认为BCG效果最好。膀胱癌在灌注治疗过程中通常存在药物作用时间短,严重影响疗效,灌注次数多等问题。本研究拟利用冻干BCG设计一种新的灌注制剂治疗膀胱肿瘤----BCG靶向缓释集成材料。该制剂将药物的靶向贴壁与缓释治疗引入到膀胱肿瘤治疗过程中,使BCG在膀胱内靶区停留时间超过48小时,可在一定程度上有效解决以上问题。Fe3O4属于尖晶石类铁氧体,其纳米粒子具备优良的超顺磁性。本文制备纳米Fe3O4采用了化学共沉淀法,并采用水浴、微波辐照、水热等方法对Fe3O4进行熟化处理,借助)CRD、TEM、VSM等方法进行测试分析。研究发现采用化学共沉淀法制备纳米Fe3O4时,Fe3+与Fe2+配比为1.75：1时最优,Fe3O4经熟化后其晶粒结晶度有所提高,颗粒尺寸在25nm以下,晶粒尺寸增大；比饱和磁化强度随熟化的温度或时间的延长而增大；纳米Fe3O4的比饱和磁化强度和结晶度经水热熟化处理后较高；纳米Fe3O4经200℃水热处理4h后,Ms可达80.0emu/g;微波辐照后的Fe3O4的Hc、Mr都较小,辐照20min后Fe3O4的Ms、Hc、Mr分别为74.6emu/g、14.5Oe、2.02emu/g。通过数据对比能够发现,化学共沉淀-微波辐照制备的Fe3O4超顺磁性较好。壳聚糖(CS)是甲壳素的脱乙酰基产物,由于其独特的生物活性、可生物降解性、生物相容性,在工业、农业、医药等领域得到广泛应用。壳聚糖-β-甘油磷酸钠(GP)温敏凝胶具有良好的组织相容性、无毒副作用、在生物体内可降解,使得CS/GP温敏水凝胶广泛应用到药剂学、组织工程等领域。壳聚糖-β-甘油磷酸钠温敏凝胶作为纳米磁性Fe3O4的载体材料,研究确定了其制备工艺参数。在壳聚糖温敏凝胶制备过程中加入纳米Fe3O4微粒,再经干燥粉碎得到Fe3O4/壳聚糖温敏凝胶磁性颗粒。SEM结果显示,由于凝胶粘度较大以及凝胶化过程中的疏水作用,Fe304产生比较严重的团聚,包覆凝胶后的Fe304颗粒粒径明显增大；XRD与IR结果显示,Fe304与凝胶基体并未发生反应；磁性能曲线表明制备的颗粒磁性能下降较大,Ms下降约80%,但颗粒仍然保持了一定的磁响应特性,且随着Fe304的含量的增加,复合颗粒的磁性能相应增大。采用分光光度计法和四唑鎓盐(XTT钠盐)法进行了BCG浓度的检测方法探究。冻干BCG为白色疏松体或粉末,复溶后为均匀悬液。其主要成份为卡介菌,辅料包括明胶、蔗糖、氯化钾、味精。XTT作为线粒体脱氢酶的作用底物,被活细胞还原成水溶性的橙黄色甲臢产物。当XTT与电子偶合剂(例如PMS)联合应用时,其所产生的水溶性的甲臢产物的吸光度与活细胞的数量成正比。实验结果表明,单纯的采用分光光度计进行BCG浓度检测,不同浓度的BCG溶液没有固定的吸收峰,无法准确的测定BCG的浓度；XTT钠盐由于其水溶性的显色反应能够反映细胞的活力,能够应用于细菌的活力检测。测定的吸光度值与配置的BCG溶液浓度呈现良好的线性比例关系,XTT方法将BCG浓度检测周期缩短为24小时,方法简单易行。在壳聚糖温敏凝胶制备过程中加入Fe304和BCG,充分搅拌分散均匀,获得载药靶向缓释复合凝胶,将凝胶高速机械粉碎得到BCG/Fe3O4/壳聚糖温敏凝胶磁性颗粒,并配制成灌注缓释液,探究BCG载药磁性复合颗粒的缓释性能。采用XTT法进行BCG缓释液浓度的检测,结果表明缓释液缓释初期在较短的时间内达到一个较高浓度,出现“突释”现象,约12小时后浓度变化逐渐趋于稳定,缓释至48小时仍能保持一定浓度,具有较好的缓释效果。由于其载药量不同,BCG药物包封率随着药物含量的增加呈现先增后降的趋势,药物含量10mg时包封率最高。基于扩散原理对缓释集成颗粒的一级方程缓释模型进行修饰,提出了靶向贴壁缓释集成颗粒的5参数对数缓释模型。对提出的5参数对数模型,以及常用的缓释模型从模拟精度、方差、预测精度等方面进行分析与比较。结果表明,构造的函数模型能准确地反映靶向载药颗粒的缓释性能。采用同样的方法制备BCG/Fe3O4/壳聚糖温敏凝胶靶向缓释颗粒,进行缓释集成颗粒靶向贴壁实验。首先在漏斗中进行体外贴壁实验,将载药靶向缓释颗粒悬浊液注入锥形漏斗,轻晃摇匀,用铷铁硼强磁体靠近漏斗侧壁,靶向磁性载药颗粒在外磁场的作用下能够聚集到漏斗侧壁上,实现可靠贴壁,漏斗内得澄清溶液；撤掉外磁场,再次轻晃漏斗,载药靶向缓释颗粒在液流作用下全部排出。其次,利用新西兰大白兔动物模型进行载药靶向缓释颗粒膀胱灌注,并通过X光及CT进行检查,可见复合材料在膀胱内部分散成堆聚集,BCG/Fe3O4/壳聚糖温敏凝胶复合颗粒贴壁效果良好。实验组按照每隔24小时处死,观察膀胱内BCG/Fe3O4/壳聚糖温敏凝胶复合颗粒情况。实验结果发现,膀胱贴壁24小时后仍有大量复合材料聚集,48小时后膀胱内材料随尿液排走较多,72小时膀胱内无材料残留。实验结果表明设计人体体外强磁体固定装置,利用强磁场作用,确保载药靶向缓释复合材料在人体膀胱内可靠贴壁,并维持48小时是切实可行和必须的。更多还原

【Abstract】 Tumor of bladder is the most common tumors of the urinary system, the vast majority of which come from epithelial tissue, and of which more than90%are transitional cell tumors. Tumor of bladder ranks fourth in men and tenth in women. The spread of the cancer heads to the bladder wall until involving the bladder tissue and adjacent organs. According to clinical habits, we refer Tis carcinoma in situ, papillary carcinoma of Ta non-infiltration and the lamina propria tumor of T1infiltration as superficial bladder cancers. General treatment of superficial bladder cancer is endoscopic laser or photodynamic therapy. To prevent the recurrence of tumor, intravesical infusion of drug can be used after treatment. The drugs commonly used are mitomycin, doxorubicin, HCPT, BCG and so on. Now it is generally considered that BCG works best. In infusion therapy process, due to the short duration of action, its efficacy is affect seriously, which result in multiple perfusion. High perfusion number and many other problems often bring patients a great deal of physical suffering and economic burden. Introducing of drug targeting adherent with sustained-release treatment bladder cancer treatment process, this study intends to design a new target sustained release integrated materials with BCG composite by freeze-dried BCG to make the BCG stay in the bladder more than48hours to attain effective solution to the above problem in a certain extent.Fe3O4belongs spinel ferrite, and its nanoparticles possess excellent superparamagnetic property. Firstly, Fe3O4nanoparticles were prepared by chemical coprecipitation. After aging treatment by heated in water bath, heated by microwave irradiation and hydrothermal, we analysis them with methods such as XRD, TEM, VSM. Using chemical coprecipitation method to prepare Fe3O4, Fe3+and Fe2+best ratio of1.75:1, the degree of crystallinity improve after curing Fe3O4grain, the grain size increases, the particle size of less than25nm; ratio of the saturation magnetization increases with the extension of the temperature or time of ripening; saturation magnetization and the degree of crystallinity of Fe3O4nanoparticles are higher after by hydrothermal aging treatment; after nano Fe3O4heat treatment at200℃water for4hours, Ms up to80.0emu/g. The He and Mr of Fe3O4treated with the microwave irradiation are small. And Ms, Hc, Mr were74.6emu/g,14.5Oe,2.02emu/g which irradiated for20min. Comparative data, we find that they have good superparamagnetic Fe3O4coprecipitation-prepared by microwave irradiation.Chitosan (CS) is deacetylated product of chitin. Because of its unique biological activity, biodegradability and biocompatibility, it has been widely used in industry, agriculture,medicine,and other fields.Chitosan-β-glycerophosphate(GP) thermosensitive hydrogel has good histocompatibility, non-toxic side effects in vivo biodegradable, which makes CS/GP temperature-sensitive hydrogels be widely applied to pharmacy, tissue engineering, and other fields. We put Chitosan-β-glycerophosphate thermo-sensitive gel as BCG and nano-magnetic ferrite carrier materials. We identified the preparation process parameters by study.90-100mg Chitosan with a deacetylation degree of95%was dissolved in4.5mL O.lmol/L of dilute hydrochloric acid, the400-500mg GP was dissolved in0.5mL of deionized water, and was added dropwise to the CS solution accompanied by stirring constantly. According to the different needs of adjusting the pH value in a37℃water bath, formation of a gel can be controlled within the time. Add the nano Fe3O4particles to the the chitosan in the thermosensitive gel preparation process, then dry and crush to get Fe3O4/chitosan thermosensitive hydrogel magnetic particles. SEM results show that, due to the gel viscosity and the hydrophobic interaction in the gelation process, Fe3O4produce more serious reunion, and the size of Fe3O4particle coated gel significantly increases. XRD and IR results show that Fe3O4didn’t react with gel matrix. Magnetic curves showed that the magnetic property of particles decline seriously, and Ms decreases by approximately80%, but the particles are still maintained a certain magnetic response characteristics and magnetic properties of the composite particles can be correspondingly increased with increasing the content of Fe3O4.Spectrophotometer method and the method of the tetrazolium salt (XTT sodium salt) are used to explore the detection method of BCG concentration. The freeze-dried BCG is white loose body powder, and uniform suspension after reconstituted. Its main components are the BCG, and its excipients include gelatin, sucrose, potassium chloride and monosodium glutamate. As the substrate of mitochondrial dehydrogenase, XTT is restored to the water-soluble orange formazan product by living cells. When XTT combines with the electronic coupling agent (such as PMS), the absorbance of water-soluble formazan product is proportional to the number of living cells. Experimental results show that simply detect BCG concentration by spectrophotometer, BCG solutions with different concentrations don’t have fixed absorption peaks. So the concentration of BCG can not be accurately measured. Due to its water-soluble color reaction, XTT sodium salt can reflect the vitality of cells, which can be applied to the vitality of detection of bacteria. The absorbance value and the BCG concentration of the solution showed a good linear proportional relationship. The XTT method will shorten the cycle of the detection of BCG concentration to24hours, which is simple.Adding Fe3O4and BCG to the chitosan thermosensitive gel in the the process of the preparation, and fully stirring to make the drug-loaded targeted sustained-release composite gel. We obtain BCG/Fe3O4/chitosan thermosensitive gel magnetic particles with high-speed mechanical crushing, and configure to release liquid perfusion. Then we explore the drug release properties of the BCG uploader magnetic composite particles. We take XTT method to detect the concentration of BCG release liquid. The results showed that in the early sustained release phase, a higher concentration can be achieved in a short period of time, and there is the "burst release" phenomenon. The concentration changes gradually stabilized after about12hours. And substantially maintaining a certain concentration to48hours, it has preferably sustained-release effect. Because of different drug loadings, the encapsulation efficiency of BCG drug first increased and then decreased with increasing drug content. When the drug content is10mg, the encapsulation efficiency is highest. Based on the diffusion theory, we modified the first class equation. And we put up with5parameters logarithmic slow-release model of targeted adherent integrated sustained release particles. For the5parameters logarithmic model as well as commonly used slow-release model, we conduct analysis and comparison from the simulation accuracy, variance, and forecast accuracy. The results show that the constructed function model can accurately reflect the release properties of the targeted drug particles.Using the same method, we prepared of BCG/Fe3O4/chitosan thermosensitive hydrogel target sustained release particles. Firstly, we conduct the vitro adherent experiments in the funnels. Inject the drug target sustained release particles suspension into the cone-shaped funnel, sway and shake. Putting NdFeB strong magnet close to the lateral wall of the funnel, we can find that the targeted magnetic drug-loaded particles can gather adherent to the side wall of the funnel. And there was clear solution in the funnel. After removed the external magnetic field, and shaked the funnel again, the targeted magnetic drug-loaded particles all discharged under the action of the flow. We take New Zealand white rabbits animal models to targeted release granules intravesical instillation, and detect by X-ray and CT. We can find that composite materials gathered in the bladder, and BCG/Fe3O4/chitosan thermosensitive hydrogel composite particles have good adherent effect. Every experimental group were put to death per24hours. Observing the BCG/Fe3O4/chitosan temperature-sensitive gel composite particles in the bladder, we can find that there are still a large number of composite material together after24hours, more material drain away with the urine after48hours, and there isn’t residual material in the bladder after72hours. The results show that the design of vitro strong magnet fixtures to ensure that the drug targeted sustained release composite particles in human bladder target adherent and maintain48hours is feasible and necessary.更多还原