【作者】 梁晓龙；

【导师】 戴志飞；

【作者基本信息】 哈尔滨工业大学 ， 生物医学仪器与工程， 2012， 博士

【摘要】 癌症是威胁人类健康和生存的重大疾病，长期以来，广大科研人员不断探索和开发各种各样的治疗方法，药物载体是其中重要的一方面，因为它可以有效地防止药物降解，将药物输送到病变区域，降低毒副作用，提高治疗效果。然而，随着时代的发展，传统药物载体逐渐暴露出了一些缺点，如稳定性差、生物相容性差、载药量低、肿瘤靶向富集少、体内循环时间短等，这些问题极大地降低了药物的生物利用度，增加了患者的痛苦，因此，迫切需要发展新型药物载体。当前，药物载体正向着可控化、智能化、绿色化和诊疗一体化的方向发展，不断涌现出了各种具有良好发展前景的新型载体。有机-无机复合载体材料就是其中的一种，它综合了有机物和无机物的特性，具备独特的优势。本文以有机无机复合材料的一种——硅质体作为研究对象，从有机-无机复合脂质分子设计的角度出发，在药物载体的结构控制释放、光控释放、光动力治疗，以及光动力治疗结合磁共振成像等方面开展了系统的研究。研究了有机无机复合脂质结构对硅质体药物释放性能的影响。通过调节脂质分子亲疏水基团的比例，合成了4种不同结构的复合脂质分子，利用溶胶凝胶和自组装技术获得了四种具有不同表层硅酸盐网络致密度的新型硅质体，并以亲水药物阿霉素和疏水药物紫杉醇为代表，成功制备了4种阿霉素硅质体和4种紫杉醇硅质体。通过体外药物释放行为和细胞毒性的对比分析，阐明了载体对药物的释放性能与相应复合脂质的结构密切相关。实验结果表明，各载体对药物均具有良好的缓释作用，当疏水基团相同时，亲水硅烷数量越多，药物的释放速率越慢；当亲水硅烷相同时，疏水基团越多，对亲水药物的释放越快，而对疏水药物的释放则越慢。细胞实验结果表明，各载药硅质体对细胞的抑制作用与其药物释放行为一致，即载体对药物的释放越快，则相同药物浓度和相同孵育时间下对细胞的抑制作用也越明显，充分体现了分子结构的设计对脂质双层渗透性的有效调控。开展了高度稳定灵敏的光响应控制释药载体材料的研究。通过有机合成的方法将光敏感基团偶氮苯与复合脂质相结合，获得了一种新型的光响应有机无机复合脂质分子，进而制备了脂质双层富含偶氮苯基团的光响应囊泡载体。通过紫外可见吸收光谱研究了囊泡中偶氮苯基团光致异构的情况，阐明了光致异构的影响因素。实验结果表明，脂质双层中偶氮苯基团与脂质双链主要以交替排列的方式分布，在紫外光和可见光轮流照射下，双层中的偶氮苯能顺利地实现可逆的构型异构，且其反-顺异构化比例可达到33.4%。以染料尼罗红作为模型药物，研究了光响应载体的光控释药性能。研究发现，在紫外光照下，载体在20min内即可释放48.2%的尼罗红，表现出灵敏的光响应控制释药能力。研制了具有光动力治疗和荧光诊断功能的硅质体。将卟啉基团引入复合脂质分子中，合成了含有双链、硅烷头部、卟啉基团的新型复合脂质，制备了相应的卟啉硅质体光动力载体材料，其光敏剂载药量可高达33.4%。通过包载亲水性染料钙黄绿素研究了卟啉硅质体的囊泡结构；通过大量的实验和讨论分析，研究了载体双层中卟啉基团的聚集和排列方式、化学共价键连卟啉基团的重要作用、单线态氧产生的情况并解释了其产生机理，探讨载体被细胞摄取的方式，并通过细胞形态变化和MTT方法研究了光动力治疗效果，在此基础上进行了初步的动物实验，考察载体在大鼠血液中的循环动力学。实验结果表明，卟啉硅质体脂质双层中卟啉基团基本不存在聚集情况，其与双链之间主要以交替方式有序排列，在紫外光照射下，载体呈现出明显的红色荧光。在重水和细胞中卟啉硅质体均能显著地产生单线态氧，且产生效率与浓度和时间成正比。激光共聚焦显微镜图片清楚地显示载体以内吞方式被肿瘤细胞摄取，且主要聚集于溶酶体中。载体对细胞表现出很低的暗毒性和显著高的光毒性，且在血液中具有长循环的特点，体现了其作为药物载体的显著优势。构建了同时具有磁共振成像与光动力治疗功能的诊疗一体化纳米粒子。在前一章合成的基础上，将卟啉与金属锰卟啉衍生物相结合，制备了一种内有双层卟啉基团外有金属锰卟啉的新型纳米粒子，其中脂质双层中的卟啉用于光动力治疗，外层的锰卟啉用于磁共振成像。研究了该类粒子的制备方法、光谱性质、单线态氧产生效率、细胞摄取实验，并在此基础上进行体外磁共振成像效果和光动力治疗效果的检测。实验结果表明，所制备的纳米粒子在透射电镜下呈现明显的核壳结构，通过调节5种不同比例的外层锰卟啉，可制备出光动力治疗和磁共振成像效果可调节的纳米粒子，随着键连锰卟啉比例的增加，粒子对水质子的纵向驰豫效率加速也越明显，达到40.1%以上时，成像效果达到最佳。细胞实验证实了该纳米粒子能被肿瘤细胞有效摄取，且对细胞具有低暗毒性和高光毒性，最终能同时满足成像和光动力治疗的纳米粒子上键连锰卟啉的最佳比例是40.1%。更多还原

【Abstract】 Cancer is a threat disease to human health and survival, for a long time, themajority of researchers have been continuing to explore and develop a variety oftreatment methods, drug delivery is an important one, because it can effectivelyprevent drug degradation, transport drug to the lesions, reduce toxicity and increasetherapeutic effect. However, with the development of the times, the traditional drugcarriers gradually revealed some shortcomings, such as poor stability, poorbiocompatibility, low drug loading efficiency, less enrichment in targeting tumor andshort circulation time in vivo, these problems greatly reduce the bioavailability ofdrug and increase the suffering of patients, therefore, there is an urgent need todevelop new drug carriers. Currently, the drug carrier is toward in the direction ofcontrolled, intelligent, green and theranostic, developing a variety of new carrierswith good prospects. Organic-inorganic hybrid material is one of them, whichcombines the characteristics of organic and inorganic materials, possessing uniqueadvantages. In this paper, an organic-inorganic hybrid materials–Cerasome wasused as the main object of study, with the view point of molecular design for theorganic-inorganic hybrid lipid, systematic research in the structure-controlledrelease, light-controlled release, photodynamic therapy, and photodynamic therapycombined with magnetic resonance imaging were carried out.The relationship between the hybrid lipid structure and drug release properties ofCerasome was researched. By adjusting the ratio of hydrophilic and hydrophobicgroups in the lipid molecules, four lipids with different structures were synthesized,later sol-gel and self-assembly technologies were applied to obtain four newCerasomes with silicate network of different surface density. Hydrophilic drugdoxorubicin and hydrophobic drug paclitaxel were used as the model drug, four kindsof doxorubicin-Cerasomes and paclitaxel-Cerasomes were successfully prepared. Invitro drug release behavior and cytotoxicity results showed that the release propertiesof drug carriers were closely related with structure of the corresponding lipids. Allthe carriers can release the drug slowly, with the same hydrophobic groups, thehigher number of hydrophilic silane group, the slower release rate of the drug. Incontrast, with the same hydrophilic silane groups, the more the hydrophobic groupresulted in the faster release rate of hydrophilic drug and the slower release for thehydrophobic drug. Cell experiments showed that the inhibition effect of the drugloaded Cerasomes were consistent with their drug release behavior, that is to say,with the same drug concentration and incubation time, the faster drug release from the carriers lead to more obvious inhibitory effect on cells. These results fully reflectthe design of the molecular structure can effectively regulate the permeability of theCerasome bilayer.Highly stable and sensitive light-responsive material for controlled drug releasehad been studied. Photosensitive azobenzene group was introduced into the hybridlipid by organic synthesis to obtained a new type of photoresponsive organic-inorganic hybrid lipid, thus light-responsive vesicle with azobenzene lipid bilayerwas prepared. Photo-isomerization of the azobenzene unit in the vesicles wasdetected by UV-visible absorption spectra, affecting factors to the photo-isomerization was clarified. The results showed that the azobenzene group anddouble-chain were mainly distributed in the vesicle bilayer with an alternative mode.With alternative irradiation by UV and visible light, the azobenzene group canachieve a reversible configuration conversion, and its trans-isomerization ratio was33.4%. Nile red was use as a model drug to study the photo controlled-releaseperformance of the carrier. The study found that upon UV light irradiation, the carriercan released Nile red of48.2%in20min, showing the sensitive capabilities of light-controlled drug release.Cerasome with fluorescence and photodynamic function was developed. Theintroduction of the porphyrin groups to the hybrid lipid resulted in a new hybrid lipidcontaining double-chain, porphyrin and silane head groups, the correspondingporphyrin Cerasome used as photodynamic agents were then prepared, its drug-loaded efficiency could reach to33.4%. The vesicular structure of porphyrinCerasome was verified by encapsulation a hydrophilic dye of calcein. A lot ofexperiments combined with discussion and analysis were carried out to study theaggregation and arrangement mode of the porphyrin groups, the important role ofchemical covalent bonded with porphyrin groups, singlet oxygen generatedefficiency and mechanism, and cellular uptaken way. Observation on cellmorphology and MTT assay were applied to test the photodynamic effect, finallypreliminary animal experiments were carried out to study the blood circulationdynamics of the carrier in rat. The results showed that double-chain and porphyrinunit should mainly arrange in an orderly alternating manner, so aggregation ofporphyrin unit in the Cerasome did not exist, upon UV light irradiation, the carriershowed brightly red fluorescence. Porphyrin Cerasome can significantly generatedsinglet oxygen in heavy water and cancer cells, and singlet oxygen generatedefficiency was proportional to the carrier concentration and light irradiating time.Confocal laser scanning microscopy images clearly showed porphyrin Cerasome wasuptaken by tumor cells through an endocytosis way, and mainly located in the lysosome. The vesicle exhibited low dark toxicity and significant phototoxicity to thecells, and it could maintain a long circulating time in the blood, showing thesignificant advantage as drug carrier.Theranostic nanoparticle simultaneously with photodynamic therapy and magneticresonance imaging abilities was design and prepared. Based on the synthesis in theprevious chapter, the porphyrin and manganese porphyrin derivatives were combinedtogether to obtain a new kind of nanoparticles with inside double-porphyrin bilayerand outside manganese porphyrin, in which inside porphyrin was for photodynamictherapy, the outer layer of manganese porphyrin was for magnetic resonance imaging.Studies for such particles were carried out, including preparation method,spectroscopic properties, singlet oxygen generated efficiency, cellular uptaken, invitro magnetic resonance imaging and photodynamic therapy testing. Experimentalresults showed that the nanoparticles showed obvious core-shell structure in TEM.By adjusting the ratio of the outer manganese porphyrins, five different nanoparticleswith adjustable photodynamic effect and magnetic resonance imaging can beprepared. The more proportion of manganese porphyrin led to the higher acceleratedlongitudinal relaxation efficiency of water proton, the ratio of higher than40.1%canresulted in the best imaging results. Cell experiments confirmed that thenanoparticles can be effectively uptaken by tumor cells and showed low dark toxicityand high phototoxicity, the ultimate nanoparticles to meet the imaging andphotodynamic therapy effect was the one with manganese porphyrins ratio of40.1%.更多还原