【作者】 王璐；

【导师】 王静；

【作者基本信息】 河北医科大学 ， 药物分析学， 2012， 硕士

【摘要】 顺铂是一种临床常用的细胞周期非特异性强效抗癌药，对于多种实体细胞瘤如黑色素瘤、头颈部癌、非小细胞肺癌等都具有抑制细胞活性作用。尽管顺铂在治疗癌症过程中能够起到很好的疗效，但其体内毒性较大，易引起严重的胃肠道反应、肾毒性、耳毒性等毒副反应而影响了临床应用。因此如何使顺铂能够选择性到达肿瘤细胞，降低药物毒性提高治疗效果是现阶段研究的主要方向。磁性热敏性脂质体是将磁性材料和药物包裹在脂质体内的新型靶向制剂，给药后在身体的局部施加固定磁场以增加其在局部滞留时间提高其靶向性，随后在温热刺激下快速释放药物起到局部杀伤效果，可减少对正常组织的影响，提高药物的治疗效果。目的：对顺铂磁性脂质体的分子组装方式进行设计，通过选用适合的磷脂前体，设计合理的制备过程，来改善脂质体的载药率，提高其靶向性。此外对二棕榈酰磷脂酰胆碱（DPPC）与多种金属离子的相互作用进行研究，探讨金属离子辅助载药的模式和机理，为提高脂质体的载药率提供新的途径。方法：采用改良水热合成方法一步制备表面被有机基团修饰的Fe3O4磁性纳米颗粒，对磁性颗粒进行XRD、TEM、IR表征。以天然蛋黄卵磷脂（EPC）为原料，采用石墨炉原子吸收分光光度法测定顺铂含量，以包封率为指标讨论磁性脂质体不同制备方法。利用薄膜分散法对影响磁性脂质体包封率的处方因素进行正交实验，确定脂质体最佳制备和处方工艺。利用EPC为磷脂材料，在筛选出来的最佳制备条件下，以顺铂抗癌药物为模型药物，探讨薄膜分散法制备脂质体过程中磁性颗粒不同的加入顺序对脂质体微观结构的影响，即程序I：将磁性颗粒先与磷脂结合再成膜制备脂质体；程序II：将磁性颗粒分散于药物溶液中水合制备脂质体。通过透射电镜对脂质体微观结构进行观察，寻找适合的纳米磁性脂质体的组装方式并对其进行体外释放实验。以DPPC为原料，分别利用两种程序制备顺铂磁性热敏性脂质体，从分子水平探讨单一组分的磷脂前体对合成的脂质体理化性质的影响，并对脂质体稳定性及药物体外释放进行质量评价。通过动物实验对磁性脂质体的磁靶向性进行研究考察。利用差热分析、红外光谱和拉曼光谱对4种金属离子(Zn²⁺，Cu²⁺，Mn²⁺，Mg²⁺)与DPPC的相互作用机理进行分析，并制备金属离子辅助载药的脂质体，探讨其提高载药率的机理。结果：改良水热法制备Fe3O4磁性纳米颗粒的方法不需要高温和氮气保护，生成的粒子粒径为8.9nm，具有超顺磁性，且表面被有机基团所修饰可提高其亲油性，增加与磷脂的结合能力。选用葡聚糖Sephadex G-50凝胶柱对磁性脂质体与游离药物进行分离。确定采用薄膜分散法制备顺铂磁性脂质体，经正交实验设计筛选了制备磁性脂质的最优处方为EPC浓度=100mg·mL^-1；EPC：CH=7：1（w/w）；EPC：Fe3O4=5：1（w/w）；顺铂浓度=1mg·mL^-1。利用EPC为磷脂材料考察两种不同制备程序对脂质体微观结构的影响，透射电镜观察发现，程序I制备的脂质体磁性颗粒分布于磷脂双层中，而程序II制备的脂质体磁性颗粒分布于脂质体中间水相。采用程序I制备的磁性脂质体在药物包封率和磁性颗粒的含量上都优于程序II，药物包封率分别为34.90±3.31%和28.34±4.72%，磁性颗粒的包封率分别为4.19±1.70和3.05±3.13mg·mL^-1，同时磁性脂质体药物包封率均高于普通脂质体。上述3种不同脂质体体外释药均符合一级释药规律，能保证一定的缓释效应。DPPC作为热敏性磷脂材料，为保证脂质体的热敏性确定了以DPPC：CH为7：1制备脂质体。程序I制备的磁性脂质体同样使磁性颗粒均匀分散在磷脂双层中，并且磁性颗粒和药物的装载量均较高分别为33.51±3.30%和2.34±0.09mg·mL^-1。与EPC相比，DPPC制备的磁性脂质体囊泡粒径较小，且均匀性和磁性颗粒在磷脂层中的分散性更好。DSC与体外热敏释放共同证明了顺铂磁性热敏性脂质体具有良好的热敏性，并且通过动物实验证明了其在体内具有明显的磁靶向，可以有效运载顺铂至靶部位。由于金属离子与DPPC的磷脂酰基团产生静电力作用改变了DPPC的构象，导致了脂质分子碳氢链排列更紧密，从而使Tm升高，并且随着金属离子原子序数的增加，半径减小，阳离子单位体积的电荷密度增加，金属离子与DPPC分子间的静电作用加强。金属离子辅助载药脂质体的药物包封率均明显高于普通脂质体，并且仍能保持着良好的热敏释药性。结论：以改良水热法制备的Fe3O4磁性纳米颗粒为磁性材料，通过先将磁性颗粒与磷脂相结合的方法制得到磁性颗粒能均匀分散在磷脂双层中，且磁性颗粒和药物的装载量均较高的磁性脂质体。同时利用热敏磷脂材料制备了具有热和磁双重靶向效果的磁性热敏性脂质体，在体内、外能保证良好的磁性靶向性。本论文中磁性脂质体的研究为靶向给药系统的研究提供了有力的借鉴。此外通过研究金属离子与DPPC相互作用为进一步探讨金属离子辅助载药的新模式奠定了一定理论基础。更多还原

【Abstract】 Cisplatin, a potent cell cycle nonspecific antineoplastic drug, is widelyused against various solid tumors, such as melanomas, head and neck tumors,endometrial cancer cell, as well as non-small cell lung cancer. Althoughtreatment with this drug is often effective, its therapeutic exploitation islimited by its severe toxicities, including nephrotoxicity, ototoxicity, as well asother side effects such as nausea and vomiting. Therefore, the significantresearches of cisplatin are selecting delivery to tumour cells, reducing drugtoxicity, and improving the therapeutic index.Thermosensitive magnetoliposomes, used as a targeted drug deliverysystem, trap magnetic nanoparticles （MNs） and drug in liposomes. Liposomestrapped with MNs allow the liposomes to be concentrated in a desired area inthe organ of a patient by magnetic force; meanwhile, the drugs could display arapid release under the local hyperthermia. Compared with ordinary liposomes,thermosensitive magnetoliposomes can play a partial destruction effects,reduce the impact of normal tissues, and improve the therapeutic effect.Objective: Assembly of cisplatin magnetic liposomes was designedusing a suitable phospholipid precursor, and a reasonable preparation processwhich could improve the drug encapsulation efficiency and targeting ofliposomes. Furthermore, the interaction of dipalmitoyl phosphatidylcholine（DPPC） with a variety of metal ions and the mechanism of the metalion-assisted cisplatin loading model were investigated, which could provide anew approach to improve drug loading efficiency.Methods:The Fe3O4MNs coating by organic functional group on surface weresynthesized by a one-step modified hydrothermal method, then, weresubjected to XRD, TEM, IR characterizations. Using egg lecithin （EPC） as phospholipid material, the magnetic liposomes were prepared by differentmethods. Using selected film dispersion method, the optimized prescription ofmagnetic liposomes was selected by orthogonal test in which encapsulationefficiency of cisplatin was used as index and graphite furnace atomicabsorption spectrophotometry （AAS） was used to determine cisplatin.Based on EPC phospholipid material, and film dispersion method,magnetic liposomes with different microstructure were prepared by the twodifferent procedures. In procedure I, MNs were combined with phospholipidsduring film formation, and in procedure II, MNs were mixed with drugsduring hydration. The microstructures of liposomes were observed bytransmission electron microscope （TEM） to seek for a reasonable assembly ofmagnetic liposomes. The release of cisplatin from liposomes was evaluated invitro system.DPPC based cisplatin thermosensitive magneticliposomes were preparedusing two different procedures. From the molecular level point of view, theinfluence of the single component phospholipid precursor on physiochemicalproperties of liposomes was investigated. The stability and drug releasecharacter of DPPC-based liposomes in vitro were evaluated, and the magnetictargeting was investigated by animal experiments. The interactions betweenfour kinds of metal ions (Zn²⁺, Cu²⁺, Mn²⁺, Mg²⁺) and DPPC were analyzed bydifferential thermal analysis （DSC）, infrared spectroscopy （IR） and Ramanspectra. Liposomes prepared using metal ions-assisted loading model and themechanism of its higher drug encapsulation efficiency were investigated.Results:The Fe3O4MNs were synthesized by a one-step modified solvothemalmethod at low temperatures and short times in a N2-free environment. Themean particle size of superparamagnetic MNs is8.9nm. The oleicacid-coating MNs could change their surface properties, improve thelipophilic character, and increase their combination ability with phospholipids.Liposomes were synthesized by film dispersion method and theencapsulation was determined using sephadex G-50column as separating instrument. The optimized prescription of magnetic liposomes was:100mg·mL^-1 of phospholipids,1mg·mL^-1 of cisplatin, EPC:CH=7:1（w/w） andEPC: Fe3O4=5:1（w/w）.The microstructures of EPC-based magnetic liposomes synthesized bythe two different procedures were observed by TEM. In procedure I, MNswere embedded in a phospholipid bilayer. In procedure II, MNs werecontained in an interior aqueous compartment. MNs-loaded liposome byprocedure I was superior over procedure II both in cisplatin encapsulationefficiency and MN content; the encapsulation efficiency of cisplain inprocedure I and II liposomes were34.90±3.31%and28.34±4.72%,respectively, while, MN content in procedure I and II liposomes were4.19±1.70and3.05±3.13mg·mL^-1respectively. Encapsulation efficiency ofcisplain in both I and II magnetic liposome were higher than MNs-freeliposome. The release profile of all the three different liposomes in vitro fittedwith a first-order equation which would ensure sustained-release character.When DPPC was used as a heat-sensitive phospholipid material,liposomes showed thermosensitive at the ratio of DPPC/CH lower than7:1.Thermosensitive magnetic liposomes prepared by procedure I also trappedwell-dispersed MNs in the bilayer; the encapsulation efficiency of cisplain andthe content of MN were33.51±3.30%and2.34±0.09mg·mL^-1, respectively.Compared with EPC-based magnetic liposomes, the mean size of liposomevesicles was smaller, and vesicles uniformity and magnetic particles dispersityin the phospholipid layer are better. Furthermore, as-prepared thermosensitivemagnetic liposomes showed fine heat sensitivity proved by DSC and in vitrothermal release experiment, meanwhile, showed the magnetic targeting in vivowhich can delivery cisplatin to the target location proved by animalexperiments.The electrostatic interation between the metal ions and phospholipid acylgroup of DPPC resulted in the change of DPPC conformation and thehydrocarbon chain of lipid molecules arranged closer than before. The TmofDPPC is increased. Followed the increasing atomic number and decreasing radius of metal ions, the charge density of cations increase, and theelectrostatic interaction between metal ions and DPPC strengthen. Theencapsulation efficiency of the metal ion-assisted loading liposomes wassignificantly higher than metal ion-free liposomes, and still maintained goodthermal release.Conclusion: The Fe3O4MNs synthesized by a one-step modifiedhydrothermal method were used to prepare magnetic liposomes. The magneticliposomes were prepared by procedure I in which combined with MNs andphospholipids during film formation. MNs were embedded in a phospholipidbilayer and the encapsulation efficiency of cisplain and the content of MNwere higher than those of conventional liposome. Thermosensitive magneticliposomes prepared by thermosensitive phospholipid material showed the dualeffect both of heat sensitivity and targeting. It would ensure sustained-releasecharacter in vitro and vivo. The investigations on magnetic liposomes in thispaper can provide a strong reference for targeted drug delivery system. Inaddition, the research on the interaction between metal ions and DPPC offereda theoretical foundation for further investigations of metal ion-assisted drugloading liposomes.更多还原