【作者】 罗永峰；

【导师】 徐军；

【作者基本信息】 广州医学院 ， 呼吸内科学， 2010， 博士

【摘要】 研究背景和目的:基因治疗,特别是最具前景的小分子RNA干扰作为基因药的治疗,选择合适的载体及导入方法是一个瓶颈,也是核酸为基础的药物能否在临床成功应用的关键。病毒载体因其具有高效转导基因的特点,是目前基因治疗临床研究的主流载体。但随着时间的推移,病毒载体介导免疫炎症以及激活原癌基因的潜在危险性等自身无法克服的缺陷已日益显现。因此,研发非病毒载体已成为基因治疗研究的热点。非病毒载体具有低毒、低免疫原性等特点,其中的壳聚糖及其衍生物因具备极高的生物相容性受到科学家们的青睐。我们在前期的研究工作中,已经研制出烷基化壳聚糖纳米核酸载体,并能成功的将靶目标核酸转入肺的上皮细胞,表达相应蛋白。但存在的缺点是转染效率低。精氨酸中的特征基团胍基能够促进细胞的内吞,具有细胞转导肽的作用,因此,对壳聚糖进行胍基化修饰有望改善原壳聚糖纳米载体转染效率低的缺点。同时,β2肾上腺能受体广泛分布于支气管平滑肌和肺组织内,依据配体-受体结合的原理,采纳临床用支气管舒张剂-β2受体兴奋剂作为归巢装置,把沙丁胺醇接枝于胍基化的壳聚糖上,有望通过靶向性修饰增加其核酸转运的效率与特异性。不仅如此,针对特定靶器官的局部给药方式具有吸收迅速、起效快、使用方便和副作用小等特点,是呼吸系统疾病治疗一种常用有效的给药途径。通过探讨超声雾化运送壳聚糖纳米核酸药的给药方式,最终构建一种新型RNA干扰壳聚糖纳米载体靶向归巢气雾治疗装置,将能极大解决RNA干扰等核酸类药物在呼吸系统疾病临床应用的问题。研究内容和方法:第一部分胍基化壳聚糖的物化性质和基因转染效率第一节胍基化壳聚糖物化性质的检测1.胍基化壳聚糖的合成2.透射电镜观察Gua-chitosan/DNA的粒径大小和分布;3.比较中性水溶液中chitosan和Gua-chitosan之间的溶解性,以及完全溶解状态下,两种载体溶液的PH值;4.凝胶阻滞实验观察两种载体的核酸结合能力;5.利用WES-8比较各种载体的细胞毒性。第二节胍基化壳聚糖转染效率的评价与机制探讨1.应用绿色荧光蛋白报告基因载体(Enhanced green fluorescent protein plasmid, pEGFP)转染293细胞,荧光显微镜观察不同壳聚糖纳米载体的转染效率,并结合流式细胞仪进行转染效率的定量分析;2.流式细胞仪检测在不同载体的包裹下单个细胞的荧光强度,了解它们对GFP表达影响;3.观察不同胍基取代度对壳聚糖转染效率的影响;4.跟踪不同时间点各种载体转染效率的变化。5.连接Pet质粒和yoyo1荧光染料,观察各种载体向细胞内转运核酸的能力。6.连接Pet质粒和yoyo1荧光染料,观察各种载体在体转运核酸的能力。第二部分载体的靶向性改造1.沙丁胺醇接枝于胍基化的壳聚糖及质谱分析。2.比较Sal-Gua-chitosan和Gua-chitosan在具有β2受体的293细胞及不表达β2受体的16HBE细胞的转染效率。第三部分超声雾化对壳聚糖核酸复合物转染效率的影响及其机理1.凝胶电泳观察超声雾化下,Gua-chitosan对于DNA的保护作用;2.分别比较载体/DNA复合物经超声雾化以后,pEGFP在293和16HBE细胞当中转染效率的变化;3.透射电镜观察超声雾化前后,载体和载体/DNA复合物粒径大小和分布的变化。结果第一部分胍基化壳聚糖的物化性质和基因转染效率第一节胍基化壳聚糖粒径在20-70nm之间;壳聚糖经胍基化修饰以后在中性水溶液当中的溶解性大大增加;胍基能降低壳聚糖自身的电荷密度,造成与DNA结合的临界复合比升高;与天然壳聚糖相仿,胍基化改造并没有增加载体的细胞毒性,两者对于细胞生长率均没有明显影响。第二节胍基化壳聚糖转染pEGFP的效率明显高于天然壳聚糖,这种转染效率的提高在一定范围内并不依赖于胍基化程度的高低。在单个细胞的水平上,胍基化壳聚糖所转染的绿色荧光蛋白的强度高于天然壳聚糖。在转染后24小时,脂质体所介导的基因表达达到峰值,而胍基化壳聚糖所介导的基因转染,蛋白表达的高峰出现在72小时。细胞体外实验和在体实验均显示,胍基化壳聚糖能更有效的地把质粒DNA运送到细胞体内。第二部分载体的靶向性改造与不表达β2受体的16HBE细胞比较,293细胞用Sal-胍基化壳聚糖纳米载体转染绿色荧光报告质粒的表达效率显著高于未引入配体的胍基化壳聚糖纳米载体。第三部分超声雾化对壳聚糖核酸复合物转染效率的影响及其机理壳聚糖能有效保护被包裹的DNA免受超声振荡的剪切作用。壳聚糖/DNA复合物经超声雾化以后转染效率显著提高。超声处理后复合物粒径分布范围较前变窄,粒径增大,颗粒均匀度较前明显改善。结论1.胍基化修饰的壳聚糖纳米载体能有效提高核酸的运送及转染效率,在生理PH值环境下能够溶于水,为临床局部应用的可行性奠定了基础。胍基化修饰的壳聚糖纳米提高转染效率的机制可能是通过提高细胞的内吞作用实现,。该特质性更有利于其在体转送核酸物质。2.β2受体兴奋剂-沙丁胺醇接枝的胍基化壳聚糖纳米载体通过引入靶向归巢装置,能够利用气道细胞存在大量β2受体的特点,依据配体-受体结合的基本原理,进一步提高细胞对核酸的摄取与转染效率,为在体经气道局部给予核酸类药奠定了重要基础。3.壳聚糖包裹能有效保护超声振荡下的DNA免受剪切破坏;同时超声雾化通过改变复合物的粒径和分布,可以有效提高核酸的转染效率。更多还原

【Abstract】 Objective and backgroundRNA interference (RNAi) is a natural cellular process that regulates gene expression by a highly precise mechanism of sequence-directed gene silencing at the stage of translation by degrading specific messenger RNAs and blocking translation. It has shown more potential than conventional gene-targeting strategies. However, the poor cellular uptake of synthetic small interfering RNA (siRNA) is a major impediment for their clinical use due to its instability, inefficient cell entry, and poor pharmacokinetic profile. Various delivery vectors have thus been developed in order to circumvent these problems. From among the gene vectors that have been studied, non-viral vectors have attracted more and more attention in comparison to viral vectors, although viral vectors have been proven to yield higher transfection efficiency in most cell lines. This is attributed to the advantages of non-viral vectors such as ease of synthesis, low immune response against the vector and unrestricted gene materials size in addition to potential benefits in terms of safety.In recent years, chitosan-based carriers are one of the non-viral vectors that have gained increasing interest for its beneficial qualities such as low toxicity, low immunogenicity, as well as an excellent biodegradability and biocompatibility. For the treatment of asthma, we previously developed a 12-alkylated chitosan nanoparticle vector that was able to deliver ECE-siRNA into airway epithelial tissues of the OVA-challenged mice, leading to down regulation of the synthesis of ET-1 to some extent. Today’s question remains the delivery of siRNAs. The 12-alkylated chitosan nanoparticle vector cannot be used in human therapy and its delivery efficiency is still the most important obstacle for the siRNA-based treatment.It has been found that the arginine-induced perturbation is preferentially ascribed to the presence of the guanidinium group in Arg, indicating that modification of chitosan with guanidinium groups would optimize its delivery efficiency for siRNA. The development of methods for specific delivery of genes into target tissues is also an critical issue for the further progress of gene therapy. Introduction of targeting ligands to nonviral vector. resulted in increased gene expression and allowed to direct transfection complexes more specifically to selected cell types in order to reduce undesired side-effects in non-target cells. The beta2-adrenergic(β2-AR) receptor is found predominantly in bronchial smooth muscle and lung tissue.β2-AR agonists may serve as a suitable targeting ligand to improve receptor-mediated gene delivery. In addition, topical delivery of the drug directly to the site of action offers the advantages of enhanced drug delivery to anatomical target site with maximum therapeutic efficacy but the minimal adverse side effect.. This has led to the widespread use of inhalation therapy in lung diseases. In present study, we developed a targeting gene carrier by covalent linkage of salbutamol to the guanidinylated chitosan(Gua-chitosan).. The effect of ultrasonic aerosol on the vector/nucleic acid complexes was also studied for topical delivery of siRNA in treatment of respiratory diseases.MethodsPart one1. Characteristics of Gua-chitosanParticle size and its distribution of the Gua-chitosan/pDNA complexes were measured by transmission electron microscopy(TEM). The water-solubility of Gua-chitosan was detected under various PH conditions. Gel retardation was evaluated for the pDNA-packaged by Gua-chitosan. The cytotoxicity of Gua-chitosan nanoparticles was tested in HEK 293 cells using CCK-8 assay.2. Transfection efficiency mediated by Gua-chitosanHEK29 cells transfected with Gua-chitosan, encapsulating the pEGFP as a reporter were observed under a fluorescence microscope. Transfection efficiency of the complexes and the mean fluorescence intensity of individual cell were further evaluated by flow cytometry. The PET plasmid was labeled with fluorescein Yoyo1 for in vitro and in vivo assessment of the transport of siRNAs across the cell membrane by Gua-chitosan nanoparticle.Part two Specific modification of Gua-chitosanSal-Gua-chitosan was synthesized by covalent linkage of salbutamol to the Gua-chitosan using epichlorohydrin. 1H NMR spectroscopy was performed on investigation of the rate of substitution of salbutamol. The transfection efficiencies of Gua-chitosan and Sal-Gua-chitosan were detected. in both HEK 293 cells and bronchial epithelial cells (16HBE).Part three Effects of Ultrasonication on the transfection efficiency mediated by the Gua-chitosanThe protection of pDNA by chitosan against ultrasonication was characterized by agarose gel retardation experiment. Transfection efficiency of the Gua-chitosan/pDNA complexes was evaluated by flow cytometry. prior to their treatment of ultrasonic aerosol.. The effects of ultrasonication on the particle diameter and size distribution of Gua-chitosan nanoparticle were determined using TEM.ResultsPart one1. In TEM images, Gua-chitosan/pDNA complexes assumed spherical particles with diameter ranging from 20nm to 70nm. Chitosan modified by guanidinium group is able to be dissolved in neutral water . In contrast, the natural chitosan is water soluble only at PH below 4.0. We show that the Gua-chitosan can effectively condense DNA in spite of a weaker DNA-binding strength relative to chitosan. Cytotoxicity Assay demonstrate that Gua-chitosan has no any negative impact on the cell proliferation.2. Expression efficiency for GFP mediated by the Gua-chitosan is greatly enhanced compared with that of chitosan-mediated transfection. The highest transfection efficiency of the both is observed 72 hours after the application of vector/pDNA complexes. The mean fluorescence intensity of GFP is higher in the cells transfected with pEGFP-Gua-chitosan nanoparticle than that in those transfected with pEGFP-chitosan. nanoparticle. The fluorescence microscope shows that Gua-chitosan vector has the capability to deliver much more Yoyo1-labled pDNA into the cells, compared to the natural chitosan. This was not only observed in vitro but also in mice submitted to intracheal instillation of the complex.Part twoOn average, about 100 glucosamine units in Gua-chitosan are successfully conjugated with 5 salbutamol molecules. The salbutamol modification of Gua-chitosan increases the expression efficiency of pEGFP in HEK293 cells that expressβ2-AR, but not in 16HBE cells withoutβ2-AR expression.Part threeThe pDNA encapsulated in Gua-chitosan is perfectly protected from a shearing force generated by ultrasonic aerosol. It is found that the Gua-chitosan nanoparticle treated by ultrasonic aerosol results in an elevated transfection efficiency. The particle sizes of Gua-chitosan and Gua-chitosan/pDNA were both increased, accompanied by a narrowing of size distribution after ultrasonication.Conclusion1. Guanidinylation modification greatly improve water-solubility of chitosan vector under the physiological PH conditiion, which is important for its clinical use. Guanidinylation modification of the chitosan accelerates the transport of -pDNA across the cell memberance by the nanoparticle vector, leading to an increase of transfection efficiency.2. Theβ2-AR agonist, salbutamol is coupled to the Gua-chitosan. This made it a homing device that increases nanoparticle’s internalization by the ligand-receptor interactions.3. The Gua-chitosna protects the DNA from being sheared by ultrasonication. On the other hand, ultrasonic aerosol increases the transfection efficiency of the Gua-chitosan by change of diameter and size distribution. We suggest that ultrasonic aerosol is an available approach for the delivery of gene vector complexes.更多还原