【作者】 刘克海；

【导师】 高申；

【作者基本信息】 第二军医大学 ， 药剂学， 2012， 博士

【摘要】 基因治疗现已成为攻克肿瘤最具希望，也是研究最为活跃的领域。基因导入系统是基因治疗的核心技术。现阶段面临的最大难题在于尚未找到理想的基因载体，治疗基因的导入仍然是肿瘤基因治疗的瓶颈。非病毒载体近年来发展迅速，其中聚乙烯亚胺(polyethylenimine PEI)是近年来研究最为广泛的阳离子多聚物非病毒基因载体，然而，聚乙烯亚胺作为基因载体使用存在三个突出问题：第一，转染效率与细胞毒性存在矛盾。小分子PEI虽细胞毒性低，但转染效果差，高分子量PEI虽具有较理想转染效率，但细胞毒性强；第二，体液环境中稳定性与穿膜能力存在矛盾。为增加复合物体液环境中的稳定性，应提高PEI/DNA复合物的亲水性，但同时穿膜能力也因而受到影响，使转导效率显著降低。第三，聚乙烯亚胺靶向性差，解决靶向性问题已成为非病毒载体中最为关注的问题。基于以上分析，本课题首先采用Pluronic P123连接低分子量聚乙烯亚胺(PEI2000)，得到多分枝状或网状结构的高分子量PEI衍生物，然后利用整合素αvβ3在人多数肿瘤细胞和肿瘤新生血管高表达的特点，选择特异亲和该整合素的RGD短肽，与细胞穿膜肽TAT(49-57)连接，合成具有靶向于αvβ3和促进载体穿膜的含RGD和TAT(49-57)双功能肽RGDC-TAT(命名为R13)，利用交联技术将R13与PEI衍生物偶联，从而构建新型非病毒基因载体系统P123-PEI-R13，并考察该系统细胞穿膜及胞内转运、释放机制。本课题针对PEI作为基因载体使用中存在的问题，旨在保证较高转染效率情况下，降低PEI细胞毒性，增加其对肿瘤细胞及其新生血管的靶向性，进而提高基因的细胞摄取水平和转染效率，提高肿瘤的基因治疗效果，为基因治疗探索一条有效的途径。本课题第一部分内容是P123-PEI-R13合成与表征。首先采用固相法制备双功能肽R13（RGDC-TAT），并通过电喷雾质谱分析鉴定其序列，通过HPLC测定其相对百分含量，通过表位肽标记的HRP来检测R13与整合素阳性的Hela细胞与B16细胞的体外结合。进而采用三光气+琥珀酰亚胺法活化Pluronic P123，并以其为反应剂交联PEI2KDa，得到高分子量PEI衍生物P123-PEI，再选择SMCC法将R13按不同反应比与P123-PEI偶联，得到目的产物P123-PEI-R13，各反应产物通过IR或1H-NMR进行结构分析。结果表明，成功合成了双功能肽R13，其序列为Arg-Gly-Asp-Cys-Arg-Lys-Lys-Aarg-Arg-Gln-Arg-Arg-Arg（RGDCRKKRRQRRR），纯度为95.8677%，同时R13体外具有亲和整合素αvβ3阳性细胞的能力；成功采用三光气+琥珀酰亚胺法活化P123并交联PEI2KDa，同时成功采用SMCC法将双功能肽R13与P123-PEI偶联，红外、核磁等结构表征表明目的产物修饰度高、纯度好，表明所选合成方法稳定、可控、重复性好。本课题第二部分内容是P123-PEI-R13理化性质。通过测定P123-PEI-R13在37℃下不同时间点分子量的方法评价其水解性能，采用琼脂糖凝胶电泳阻滞分析考察其缩合DNA能力以及对质粒DNA抗DNase I、FBS和肝素钠酶解及解离能力，利用透射电镜观察复合物形态，使用激光粒度仪和zeta电位仪测定粒径、电位，采用MTT法评价合成的P123-PEI-R13对Hela细胞、B16细胞的毒性，并与PEI25kDa对照。结果表明，聚合物P123-PEI-R13在37℃可缓慢水解，大约60h左右可水解成小分子化合物，其水解过程可用一级动力学方程描述。P123-PEI-R13/DNA复合物呈近似球形的胶束样结构，粒径在100-300nm之间，电位适中。P123-PEI-R13与DNA质量比为2时能与DNA完全结合，同时可抵抗高达280μg/mL的肝素钠、50%的FBS及6UDNase I/DNA的解离或酶解。相比较PEI25kDa，P123-PEI-R13细胞毒性显著降低。本课题第三部分内容是P123-PEI-R13/DNA纳米复合物体内外转染。以绿色荧光蛋白质粒pEGFP-N2和虫荧光素酶质粒pGL3-Control作为报告基因，评价其对Hela人宫颈癌细胞、B16小鼠黑色素瘤细胞及HepG2人肝癌细胞转染能力，采用流式细胞仪和发光仪测定绿色荧光蛋白表达阳性细胞百分率和转染细胞的虫荧光素酶活性，分别定性定量考察P123-PEI-R13对各受试细胞体外转染效率，同时，构建肿瘤模型，以虫荧光素酶质粒pGL3-Control作为报告基因，评价P123-PEI-R13递送DNA时在体内的分布与转染效率。体外转染试验表明，在考察的w/w范围内，随w/w比提高，转染效率有逐渐增强的趋势；随R13修饰比提高，转染效率有逐渐下降趋势；整体而言，P123-PEI-R13在各细胞转染远好于PEI2KDa，也好于P123-PEI，比PEI25KDa略强或近似。体内转染试验表明，P123-PEI-R13在HepG2人肝癌移植模型与B16小鼠黑色素瘤移植模型转染情况类似，整体转染能力较P123-PEI与PEI25KDa强，同时也改善了复合物的体内分布，并且由于R13的修饰，使其在肿瘤组织转染效率显著提高，可知聚合物P123-PEI-R13在体内有较强的肿瘤靶向能力，但与其对二种肿瘤细胞的体外转染相比效率要低。本课题第四部分内容是P123-PEI-R13/DNA纳米复合物穿膜及胞内转运、释放机制的研究。本部分采用加入不同内吞途径抑制剂对转染效率是否产生影响的方法，考察复合物内吞途径；采用加入内涵体-溶酶体酸化抑制剂、细胞骨架和动力蛋白抑制剂对转染效率是否产生影响的方法，考察复合物细胞内转运机制；采用细胞因子及细胞内环境是否影响复合物稳定性的方法，考察复合物在细胞内的解离；采用荧光标记聚合物并结合激光共聚焦显微镜考察复合物在细胞内的实时定位。结果表明，P123-PEI-R13修饰R13后，使其细胞摄取表现出了不同特性，P123-PEI-R13/DNA复合物可通过网格蛋白介导的内吞、小窝蛋白介导的内吞及巨胞饮三条途径内吞入胞，并可能有不依赖能量的非内吞途径存在。P123-PEI-R13/DNA复合物入胞后首先经内涵体-溶酶体系统酸化过程而从中逃逸，并以微管为轨道和方向，以动力蛋白为动力来源，在微丝作用下，并有中间纤维协助，向微管“-”端，即细胞核方向转运。RNA与P123-PEI-R13具有更强亲和性，P123-PEI-R13/DNA复合物在细胞核内被RNA解离，从而释放DNA。本课题通过Pluronic P123连接低分子量PEI制备高分子量PEI衍生物，并利用交联技术将双功能R13与该衍生物偶联，研制新型具有主动靶向αvβ3作用并且具有高转染效率、低毒性的阳离子聚合物/DNA纳米复合物，并探讨其细胞穿膜及胞内转运、释放机制，为解决目前基因给药方式存在的问题和实际应用打下基础，本课题研究内容有着一定的理论意义和实践应用价值。更多还原

【Abstract】 Gene therapy provides a promising tool to eradicate cancer by treating it at its source.The key to gene therapy is finding an ideal gene delivery vector. Delivery of nucleic acidsinto cells using cationic polymers has recently gained a remarkable interest in the field ofnon-viral gene therapy due to their structural diversity, easy production,non-immunogenicity and safety. One of the most effective and widely studied syntheticnon-viral gene delivery vectors is the polycation polyethylenimine (PEI). However,polyplexes of PEI/DNA have shown three outstanding problems. First of all, long PEIchains are highly effective in gene transfection, but more cytotoxic. Conversely, short PEIchains display lower cytotoxic, but lower efficient. And then, there is a contradictionbetween stability and cell penetrating. To increase the stability of PEI/DNA complexes,their hydrophilicity should be improved. But cell penetrating is affected accordingly andtransfection efficiency decreases. At last, the major drawback with gene delivery using PEIis the lack of satisfactory specificity towards tumor cells because there is no bindingselectivity between the positively charged polycation and the negatively charged bodycells.Above all, in this work at first we synthesized a kind of high molecular weight PEIderivate (P123-PEI) by cross-linking low molecular weight PEI with Pluronic P123. Theαvβ3receptors were highly expressed on tumor cells and tumor angiogenic blood vessels,whereas they were not detectable on quiescent blood vessels. Then we used αvβ3-targetingpeptide RGD, in conjunction with the cell-penetrating peptide TAT to yield a bifunctionalpeptide RGD-TAT named R13which can improve cell selection and increase cellularuptake, and at last adopted R13to modify P123-PEI so as to prepare a new polymeric genevector (P123-PEI-R13). Mechanisms of cellular uptake, subsequent intracellular traffickingand disassembly of this vector were also investigated. The purpose of the present studywas to solve the efficiency-versus-cytotoxicity and tumor-targeting problems of PEI usedas a non-viral gene delivery vector. The new non-viral gene vector P123-PEI-R13couldreduce cytotoxicity of PEI on the premise of ensuring higher transfection efficiency,improve its tumor targeting, increase cellular uptake of gene, and then enhance thetherapeutic effect of gene therapy on cancer. This study could find an available way togene therapy on cancer. The first part of this work was synthesis and characterizations of P123-PEI-R13. abifunctional peptide R13（RGDC-TAT） was prepared using the solid phase method atfirst. The sequence analysis, content assaying and binding assessment of R13to αvβ3positive cells (Hela cells and B16cells) were performed by ESI-MS, HPLC and HRPlabeling respectively. Then we activated Pluronic P123withbis-(trichloromethyl)-carbonate and solid N-hydroxysuccinimide and employed theactivated P123to crosslink PEI2KDa so as to prepare a high molecular weight PEIderivate of P123-PEI. At last R13was used to modify P123-PEI by SMCC andP123-PEI-R13was obtained. All reaction products were characterized by IR or1H-NMR.The results indicated that the bifunctional peptide R13was synthesized successfully withsequence of RGDCRKKRRQRRR, content of95.8677%and ability of binding to αvβ3positive cells in vitro. P123has been activated and P123-PEI has been also prepared bycross-linking PEI2KDa with the activated P123. The bifunctional peptide R13wascoupled with P123-PEI by SMCC successfully. Structural analysis of IR and1H-NMRrevealed the high modification degree and purity of the products, which showed that thesynthesis method had stability, controllability and repeatability.In the second part, the new synthesized gene vector P123-PEI-R13was characterized interms of its physico-chemical properties. Degradation of P123-PEI-R13was estimated bythe measurement of molecular weight. The sizes and zeta potential of polymer/DNAcomplexes in PBS buffer at room temperature were measured using an electrophoretic lightscattering spectrophotometer. The DNA condensation capacity, the resistance to DNase Idigestion and the resistance to FBS, sodium heparin disassembly of P123-PEI-R13wasdetermined by gel retardation experiments. The cytotoxicity of the polymers on the B16and Hela cells were measured using the MTT assay in comparison with PEI25kDa. Theresults indicated that P123-PEI-R13was degraded slowly and the degradation was nearlycompleted after about60h. The degradation profile of P123-PEI-R13could be welldescribed by first-order model. The particle size of P123-PEI-R13/DNA complexes wasaround100-250nm, with proper zeta potential. P123-PEI-R13was able to condense DNAeffectively and neutralized its charge at w/w ratio of2. The nanoparticles can protectplasmid DNA from being digested by DNase I at a concentration of6U DNase I/μg DNA.The nanoparticles were resistant to dissociation induced by50%fetal bovine serum and600μg/mL sodium heparin. P123-PEI-R13showed significantly higher cell viability ascompared with PEI25kDa. In the third part, gene transfection of this vector was investigated in vitro and in vivo.We examined the ability of P123-PEI-R13to transfect Hela cells, B16cells and HepG2cells using the plasmid pEGFP-N2and pGL3-Control. Percentage of GFP transfection andluciferase activity were respectively determined using flow cytometry and a luminometerso that the quantitative and qualitative study on in vitro transfection ofP123-PEI-R13/DNA complexes could be carried out. In addition, we established animaltumor model and investigated distribution and transfection efficiency ofP123-PEI-R13/DNA complexes using the plasmid pGL3-Control in vivo. The transfectionexperiments in vitro showed that transfection efficiency of P123-PEI-R13/DNA complexesincreased in correlation with the w/w ratio and decreased with R13modification degree.On a whole, all synthesized P123-PEI-R13showed much higher gene transfer abilitycompared with PEI2KDa, also higher than P123-PEI and similar to PEI2KDa, evenhigher. The transfection experiment in vivo showed that transfection of P123-PEI-R13inthe HepG2liver tumor model was similar to that in the B16melanoma tumor model.P123-PEI-R13showed higher gene transfer ability compared with P123-PEI and PEI25KDa, ameliorated the distribution of the complexes and improved the transfectionefficiency in tumor tissues significantly due to the produce of R13. This showed thepolymer P123-PEI-R13had tumor targeting in vivo. However, the transfection efficiencyof pGL3-Control in viv was lower than that in vitro.The last part of this work was the mechanisms of cellular uptake, subsequentintracellular trafficking and disassembly of this vector. The internalization pathways ofP123-PEI-R13/DNA complexes were investigated based on the effect of specific endocyticinhibitors on transfection efficiency. The mechanism of intracellular trafficking wasinvestigated based on the effect of endosome-lysosome acidification inhibitors,cytoskeleton and dynein inhibitors on transfection efficiency. The intracellular disassemblyof P123-PEI-R13/DNA complexes was also investigated based on the effect of cytokineand cellular environment on stability of polyplexes. Intracellular localization ofFITC-labeled P123-PEI-R13polyplexes in Hela cells was researched in order to furtherclarify the transport process in the cells. The results indicated that the modification ofP123-PEI-R13with R13made it display new property of internalization.P123-PEI-R13/DNA complexes were endocyzed by clathrin-mediated endocytosis,caveolin-mediated endocytosis, macropinocytosis and possible energy-independent route.After internalization, P123-PEI-R13/DNA complexes could escape from the endosome-lysosome system because of its acidification and further took microtubule as thetrack, dynein as the dynamic source to transport towards the microtubule (+) end, to witnucleus, under the action of microfilament and with the aid of intermediate filament.P123-PEI-R13had a higher affinity for RNA. It was RNA that resulted in the disassemblyof P123-PEI-R13/DNA complexes in the nucleus.We developed a new non-viral gene vector, PEI-P123-R13, by cross-linking lowmolecular weight PEI with P123and further coupling a bifunctional peptide R13to thepolymer for targeting tumor and increasing cellular uptake. This new polymer might be apotential candidate in gene delivery with low cytotoxicity and high transfection efficiency.Also, the mechanisms of cellular uptake, subsequent intracellular trafficking and disassemblyof this vector were investigated. This study can form the base of problems solving andpractical applications of PEI as a non-viral gene delivery vector. The contents of this workdisplay theoretical as well as practical values.更多还原