基于热敏脂质体及低密度脂蛋白模型的纳米载体靶向药物递送系统研究

发布时间：2018-01-04 22:32

本文关键词：基于热敏脂质体及低密度脂蛋白模型的纳米载体靶向药物递送系统研究　出处：《中国人民解放军军事医学科学院》2017年博士论文　论文类型：学位论文

【摘要】：背景在肿瘤治疗领域,主动靶向药物递送系统早已引起各国的广泛关注。虽然该研究方向的论文发表量呈指数增加,却基本没有上市产品。基于物理化学和生物学原理分析,通过思考分子与纳米粒子在水介质中扩散能力的差异,以及纳米载体在体内的寻靶过程,已有很多研究工作剖析了主动靶向药物递送系统理论中存在的某些误区,揭示出主动靶向修饰的纳米载体有时并不能够按照载体设计初衷提高对肿瘤组织靶向效率的缺陷。EPR效应只适用于药物分子和具有足够扩散能力的纳米载体。目的为真正提高纳米载体对于肿瘤组织的靶向效率,提高成药性,我们将目光聚焦于依靠环境特异性响应的靶向释药纳米载体(以热敏感脂质体为模型)和具有强扩散能力的主动靶向纳米载体(以低密度脂蛋白为模型),这两种最具有可行性的靶向策略。本研究中旨在研究制备长春新碱及阿霉素共载药热敏脂质体(VD-TSL)和长春新碱人工低密度脂蛋白(VCR-a LDL),并进行一系列的物理化学性质评价,体外细胞评价,以及体内活体成像、药效学研究,说明这两种靶向策略可以显著提高纳米载体的肿瘤组织靶向效率。内容与方法第一部分:首先建立VCR与DOX同步分析的含量测定液相色谱检测方法,并进行了一系列方法学验证,接着采用薄膜分散法制备空白脂质体,p H梯度主动载药法实现VCR和DOX的同步载药,制备得到VD-TSL。以形态学研究、粒径和zeta电位、外观、热敏特性、包封率、磷脂分析、稳定性等为主要评价指标,进行评价。通过MTT法测定各组制剂结合加热对于两个细胞系的毒性,并利用激光共聚焦显微镜观察细胞摄取情况。建立裸鼠异种移植肿瘤模型,结合体外肿瘤部位局部加热,对VD-TSL进行了活体成像体内靶向分布研究,并对药效学及毒性进行了初步评价。第二部分:首先研究处方工艺,利用薄膜分散一步载药法,制备100nm和35nm的VCR-a LDL,随之进行粒径、zeta电位、包封率、形态学研究及稳定性等一系列物理化学性质评价。为比较两种粒径VCR-a LDL的扩散能力,我们进行了MTT单层细胞毒性研究、MTT扩散倒置细胞毒性研究、激光共聚焦观察肿瘤饼和肿瘤球模型载体渗透作用实验。接着建立了裸鼠异种移植肿瘤模型,进行VCR-a LDL的3D活体成像实验、组织分布研究及药效学评价。结果第一部分:制备得到平均粒径在85nm左右的VD-TSL,zeta电位约为零;透射电镜下观察,外观圆整;包封率达95%以上;制备多批样品,处方工艺重现性好;-20℃条件下放置6个月,VD-TSL稳定性良好。体外细胞毒性评价和摄取实验均表明VCR与DOX具有协同作用,同时加热还可以促进热敏脂质体进入肿瘤细胞内部,即包载药物的热敏脂质体与加热也有协同增效的作用。裸鼠异种移植模型活体成像实验也证明了以上结论。药效学实验结果表明,VDTSL结合肿瘤组织局部加热,具有很好的肿瘤抑制能力,抑瘤率达到了70%以上。同时,VD-TSL组裸鼠活动能力优于注射液组,且体重下降幅度最小,即显著降低了裸鼠的全身毒性。第二部分:制备了两个不同粒径的具有脂溶性核心和亲水性外壳结构的长春新碱人工低密度脂蛋白(VCR-a LDL),其中间粒径分别为100nm左右和35nm左右,呈正态分布,zeta电位基本为零。透射电镜下观察,可见VCR-a LDL的粒径大小分布均匀,外观圆整,并具有明显的壳核结构。不论100nm还是35nm VCR-a LDL,药物包封率均能够达到95%以上,且稳定性良好。细胞毒性MTT实验中两种粒径VCR-a LDL的细胞抑制率并无显著性差异,但MTT扩散倒置细胞毒性研究中,35nm VCR-a LDL扩散能力明显强于100nm VCR-a LDL,细胞抑制率更强。激光共聚焦显微镜下观察荧光标记的不同粒径a LDL对于体外肿瘤饼和肿瘤球模型的渗透作用,可见35nm a LDL的渗透能力虽没有荧光分子强,但显著强于100nm a LDL。3D活体成像实验中,与注射液组和100nm VCR-a LDL组相比,35nm VCR-a LDL组的肿瘤组织靶向滞留作用最强。药效学实验中,35nm VCR-a LDL组肿瘤抑制率最强,同时裸小鼠体重下降幅度最小,全身毒性最小。结论本研究成功研发制备了VD-TSL和VCR-a LDL,二者具有不同的靶向肿瘤释药原理,同时均具有良好的体外物理化学性质,及体内肿瘤组织靶向作用。工艺稳定重现性好。VD-TSL中结合了VCR与DOX的协同作用,以及载药热敏脂质体与加热效应的协同作用,是具有双重协同作用的环境控制靶向载体。VCR-a LDL中,a LDL本身自带主动靶向肿瘤的功能,通过考察100nm和35nm a LDL的靶向作用差异,说明减小粒径可以提高其扩散能力,能够充分利用EPR效应实现肿瘤组织被动靶向作用,才有基础进一步发挥主动靶向作用。
[Abstract]:The background in the field of cancer treatment, attention of active targeting drug delivery system has caused the world. Although the research papers has increased exponentially, but basically no listed products. Analysis of physical chemistry and biology based on the principle of differential diffusion ability in aqueous medium by thinking of molecules and nanoparticles, and the nanoparticles in in the targeting process, there has been a lot of research work on active targeting drug delivery system of certain errors in the theory, reveals the nano carrier to the modified active target is sometimes not according to the original design to improve the carrier for tumor targeting efficiency defects.EPR effect is only applicable to drug molecules and nanoparticles with sufficient diffusion capacity objective. To truly improve the nano carrier for tumor targeting efficiency, improve the resistance, we will focus on relying on the environment Drug targeting nano carrier specific response (by thermosensitive liposomes as model) and has strong diffusion ability of active targeting nanoparticles (with low density lipoprotein as a model of the two), the most feasible targeting strategy. Study on Preparation of vincristine and adriamycin were loaded thermosensitive liposomes to in this study, vincristine (VD-TSL) and artificial low density lipoprotein (VCR-a LDL), and evaluate the physical and chemical properties of a series of in vitro and in vivo evaluation, in vivo imaging, pharmacodynamics, which indicates that these two kinds of targeting strategies can significantly improve the tumor tissue to target nanoparticles content and method of efficiency. The first part: first establish content synchronization analysis of VCR and DOX were determined by liquid chromatography detection method, and conducted a series of method validation, then using blank liposomes, P H gradient active loading method VCR Synchronous loading and DOX, VD-TSL. was prepared by morphology, particle size and zeta potential, appearance, thermal properties, encapsulation efficiency, stability of phospholipid analysis, evaluation as the main evaluation index. Through the MTT method for the determination of each preparation combined with toxic heating for two cells, and the cells were observed using the uptake of confocal laser scanning microscopy. The establishment of nude mouse xenograft tumor model, combined with in vitro tumor local heating, VD-TSL for in vivo imaging in vivo targeting Distribution Research of pharmacodynamics and toxicity were evaluated. The second part: firstly, the formulation process, dispersion step loading method using thin film preparation. 100nm 35nM and VCR-a LDL, along with the particle size, zeta potential, encapsulation efficiency, stability and evaluation of morphological study and a series of physical and chemical properties. For the comparison of two kinds of particle diffusion diameter VCR-a LDL, we are MTT Study on monolayer cell toxicity, cytotoxicity of inverted MTT diffusion, laser confocal observation of tumor and tumor cake ball model carrier penetration experiment. Then we established nude mice xenograft model, 3D imaging experiments for VCR-a LDL, the study on tissue distribution and pharmacodynamic evaluation. Results the first part: the preparation of the average particle size about 85nm VD-TSL, the zeta potential is approximately zero; TEM round appearance; the entrapment efficiency was above 95%; the preparation of samples, prescription and process reproducibility; for 6 months at -20 DEG C, VD-TSL stability is good. In vitro cytotoxicity and uptake experiments showed that VCR and DOX has a synergistic effect, but also can promote the heat sensitive liposomes into tumor cells, namely thermosensitive liposomes and heating ofencapsulated drugs have synergistic effect. A nude mouse xenograft model in vivo imaging experiment Also prove the above conclusion. Pharmacodynamics experimental results show that VDTSL combined with local heating of tumor tissue, have good tumor inhibition ability, the inhibition rate reached more than 70%. At the same time, the mice activity of VD-TSL is better than that of injection group, and the weight of the smallest decline, which significantly reduce the systemic toxicity of nude mice. The second part: the preparation of two different sizes with fat soluble vincristine artificial LDL core and a hydrophilic shell structure (VCR-a LDL), the particle sizes were 100nm and 35nM, normal distribution, zeta potential is zero. Transmission electron microscope, visible VCR-a LDL particle size distribution, roundness appearance, and has obvious core-shell structure. Both 100nm and 35nM VCR-a LDL, the drug entrapment rate can reach more than 95%, and good stability. The cytotoxicity of MTT in experiment two grain size VCR-a LDL There is no significant difference between the inhibition rate of cells, but the cytotoxicity in MTT diffusion of inverted 35nM, VCR-a LDL diffusion capacity is better than 100nm VCR-a LDL, cell inhibition rate is stronger. Under a laser scanning confocal microscope and fluorescence labeled with different sizes of a LDL for in vitro tumor infiltration and tumor cake ball model, penetration of visible 35nM a LDL has no strong fluorescence, but was significantly stronger than 100nm a LDL.3D in vivo imaging experiments, and the injection group compared with 100nm group LDL VCR-a, 35nM VCR-a LDL tumor target group to stay the strongest effect. The pharmacodynamics experiment, 35nM VCR-a LDL the tumor inhibition rate and the strongest, decrease of body weight of nude mice the smallest, and minimal systemic toxicity. Conclusion the successful development of VD-TSL and VCR-a LDL were prepared by two different tumor targeting drug release in vitro and principle, have good physical and chemical properties, And in vivo tumor targeting effect. Combined with the stable process to reproduce the synergetic effect of VCR and DOX of.VD-TSL, and the drug loaded thermosensitive liposomes and the heating effect is synergistic with dual synergistic effects of environmental control targeting carrier.VCR-a LDL, a LDL has active tumor targeting function the difference to 100nm and 35nM a by investigating the role of LDL target, indicating decreasing particle size can improve the diffusion ability, can realize the passive targeting of tumor tissue to make full use of the EPR effect, only the foundation of further play an active role in targeting.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R943

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