改性聚乙烯亚胺及其叶酸修饰的反义寡核苷酸传递系统的研究

发布时间：2018-07-05 12:28

本文选题：聚乙烯亚胺 + 疏水性修饰　；参考：《吉林大学》2016年博士论文

【摘要】：在肿瘤治疗领域中,反义寡核苷酸(Antisense oligonucleotide,AS-ODN)类药物及相关治疗技术越来越得到大家的重视。目前虽然已有AS-ODN类药物进入临床阶段,但仍存在稳定性差、跨膜能力弱、易被核酸酶降解等问题。LOR-2501是一种含有20个碱基的AS-ODN,因为能够与核糖核苷酸还原酶(Ribonucleotide reductase,RNR)大亚基R1的m RNA编码区互补,具有一定的抗肿瘤活性。目前大量的研究集中于AS-ODN类药物载体的开发和应用领域。聚乙烯亚胺(Polyethyleneimine,PEI)与其他阳离子聚合物相比,有着独特的“质子海绵效应”,PEI装载药物后能够通过大量的氨基吸收质子,导致溶酶体渗透性肿胀,破裂后将药物释放到细胞质中。PEI作为AS-ODN载体时,高分子量的PEI(25k Da)转染效率相对较高但是毒性较大,而低分子量的PEI(800 Da)毒性较小但转染效率也相对较低,因此如何通过PEI的改性和靶向配体修饰等研究方法在提高PEI转染效率的同时减少其毒性成为了研究热点。本论文研究内容主要包括以下几部分:1.PEI-SS-OA的合成及载药系统的建立本文首先在PEI的基础上,合成了一种含有二硫键(S-S)的聚合物,使用油胺(Oleylamine,OA)对其进行了疏水性修饰(命名为PEI-SS-OA)。同时采用乙醇注入法制备了PEI-SS-OA载药系统。制备的PEI-SS-OA载药系统具有一定的缓冲容量,进入细胞后能够有效地释放自身携带的药物。在最优制备条件下的粒径为201.0±5.9 nm,zeta电位值为43.5±0.9 m V。PEI-SS-OA对He La和A549细胞无明显毒性,细胞活力均在90%以上。在30天内,PEI-SS-OA载药系统的粒径略有增加,zeta电位值无明显变化。2.PEI-SS-OA/LOR-2501传递系统的建立和评价为了对制备的PEI-SS-OA载体的转染效率进行评价,本文选择了一种AS-ODN类药物LOR-2501,建立了PEI-SS-OA/LOR-2501传递系统。首先以粒径和zeta电位为指标,得到了最佳的氮磷比(N/P)为8:1。此时,PEI-SS-OA与LOR-2501已完全结合,还原剂的加入能够促使LOR-2501的释放。体外细胞评价实验中,MTT抗肿瘤实验和流式细胞计数法测定转染效率实验结果表明制备的PEI-SS-OA载体能够将LOR-2501转染进入到He La和A549细胞中,抑制肿瘤细胞的生长。采用激光共聚焦显微镜观察到PEI-SS-OA/LOR-2501复合物能够大量地进入到肿瘤细胞。实时荧光定量PCR测定R1 m RNA结果显示,复合物在He La和A549细胞中对R1 m RNA的下调效率分别为40.8%和43.6%,与PEI相比分别提高了17.8%和23.3%。Western-blot测定R1蛋白结果显示,PEI-SS-OA组的R1蛋白下调效率明显高于PEI组。3.PEI-NH-OA的合成及载药系统的建立本文同时在PEI的基础上,合成了一种含有酰胺键(N-H)的聚合物,使用油酰(Oleoacyl,OA)对其进行了疏水性修饰(命名为PEI-NH-OA)。采用乙醇注入法制备了PEI-NH-OA载药系统。PEI-NH-OA同样具有一定的缓冲容量。在最优制备条件下的粒径为102.0±3.9 nm,zeta电位值为54.5±3.1 m V。PEI-NH-OA与PEI本身相比毒性明显减小,He La和A549细胞活力均在90%以上。在30天内,PEI-NH-OA载药系统粒径和zeta电位均无明显变化。与PEI-SS-OA载药系统相比,PEI-NH-OA载药系统粒径更小,阳离子特性更强,具有更好的稳定性。4.PEI-NH-OA/LOR-2501传递系统的建立和评价本文同时建立了PEI-NH-OA/LOR-2501传递系统,对PEI-NH-OA的转染效率进行了评价。以粒径和zeta电位为指标,得到了最佳的N/P为6:1。此时,PEI-NH-OA与LOR-2501已完全结合,形成纳米复合物。抗肿瘤实验显示复合物对He La和A549细胞的抑制率分别为46.2%和41.5%。通过流式细胞计数和激光共聚焦显微镜观察发现,复合物能够大量地进入到He La和A549肿瘤细胞内。实时荧光定量PCR测定R1 m RNA结果显示,PEI-NH-OA作为载体时,在He La和A549细胞中对R1 m RNA的下调效率分别为37.7%和34.5%,而PEI组仅为20%左右。Western-blot测定R1蛋白结果显示,R1蛋白的下调率分别为50.1%和46.3%。可见PEI-NH-OA能够高效地将LOR-2501转染进入细胞,与PEI-SS-OA相比稳定性较高且粒径较小,后期实验中选择对其进行进一步修饰。5.FA/PEI-NH-OA/LOR-2501传递系统的建立和评价本文在PEI-NH-OA/LOR-2501复合物基础上,进一步将叶酸(Folic acid,FA)与上述复合物通过静电吸附作用结合,得到FA/PEI-NH-OA/LOR-2501传递系统。FA/PEI-NH-OA/LOR-2501复合物在最佳比例时粒径为159.0±1.1 nm,zeta电位值为-12.2±1.3 m V,加入FA后的PEI-NH-OA载体仍能够与LOR-2501紧密结合。FA/PEI-NH-OA载体无明显毒性,细胞活力均在80%以上。体外细胞抗肿瘤评价实验中,对He La和A549细胞的抑制率分别达到55.6%和53.7%。流式细胞计数检测转染效率结果显示,复合物在叶酸受体高表达(FR+)的He La细胞中和叶酸受体低表达(FR-)的A549细胞中,平均荧光强度是FA修饰前的2倍以上,在KB细胞(FR+)和SK-HEP-1细胞(FR-)中同样出现此现象。激光共聚焦显微镜观察结果显示复合物能够大量地进入到肿瘤细胞中。R1 m RNA测定结果显示,在He La和A549细胞中,R1 m RNA下调效率分别为51.7%和45.7%。Western-blot测定R1蛋白结果显示,R1蛋白下调效率分别达到72.0%和68.9%,与未修饰的载体相比大幅提高。6.FA/PEI-NH-OA/LOR-2501传递系统内化机制的初步研究首先考察了游离FA对FA/PEI-NH-OA/LOR-2501纳米复合物进入肿瘤细胞过程的影响。结果显示,加入浓度为0.01、0.1、1 mmol/L的游离FA组与未加入游离FA组相比,无论在He La、KB细胞(FR+)还是A549、SK-HEP-1细胞(FR-)中,并没有对复合物的摄取情况有一定的影响。同时考察了三种内吞途径抑制剂蔗糖、细胞松弛素D和制霉菌素对复合物内吞过程的影响。结果显示,复合物可能主要是通过网格蛋白介导的内吞途径进入到细胞中的。使用三种内化途径特异性染料标记细胞后,采用激光共聚焦显微镜观察复合物的摄取情况,结果进一步验证了复合物主要是通过网格蛋白介导的内吞途径进入到肿瘤细胞的。综上所述,本文中制备的疏水改性的PEI及其FA修饰的载药系统具有低毒高效的特点,FA/PEI-NH-OA载药系统的转染效率与肿瘤细胞表面的FR表达量无关,在多种肿瘤细胞系中均具有较高的转染效率,有望成为安全高效的AS-ODN类药物载体。
[Abstract]:In the field of cancer treatment, antisense oligodeoxynucleotides (Antisense oligonucleotide, AS-ODN) drugs and related therapies have attracted more and more attention. Although AS-ODN drugs have entered the clinical stage, there are still poor stability, weak transmembrane ability and easy to be degraded by nucleoacid,.LOR-2501 is a kind of 20 bases. AS-ODN, because it is complementary to the m RNA coding region of the Ribonucleotide reductase, RNR, R1, and has a certain anti-tumor activity. A great deal of research is focused on the development and application of AS-ODN drug carriers. The special "proton sponge effect", after PEI loading drugs, can absorb protons through a large number of amino groups, resulting in the osmotic swelling of the lysosome. When the drug is released to the cytoplasm and.PEI as a AS-ODN carrier after rupture, the high molecular weight PEI (25K Da) transfection efficiency is relatively high but the toxicity is larger, while the low molecular weight PEI (800 Da) is less toxic but less toxic but less toxic. The transfection efficiency is also relatively low, so how to improve the efficiency of PEI transfection and reduce its toxicity through the modification of PEI and targeting ligand modification has become a hot topic. The main contents of this paper include the following parts: the synthesis of 1.PEI-SS-OA and the establishment of the drug loading system, based on the PEI, were synthesized. A polymer containing two sulfur bonds (S-S) is hydrophobic modified (named PEI-SS-OA) using Oleylamine (OA). At the same time, the PEI-SS-OA drug delivery system is prepared by ethanol injection. The prepared PEI-SS-OA drug loading system has a certain buffer capacity and can effectively release its own drug after entering the cell. The particle size of the optimal preparation was 201 + 5.9 nm, the zeta potential value was 43.5 + 0.9 m V.PEI-SS-OA for He La and A549 cells, and the cell viability was above 90%. In 30 days, the particle size of the PEI-SS-OA carrier system increased slightly and the zeta potential value had no obvious change, and the.2.PEI-SS-OA/ LOR-2501 transmission system was established and evaluated for the preparation and evaluation of the.2.PEI-SS-OA/ LOR-2501 transmission system. The transfection efficiency of the PEI-SS-OA carrier was evaluated. In this paper, a kind of AS-ODN drug LOR-2501 was selected and the PEI-SS-OA/LOR-2501 transmission system was established. The optimum nitrogen and phosphorus ratio (N/P) was obtained by using the particle size and zeta potential as the index. At this time, PEI-SS-OA and LOR-2501 had been completely combined, and the addition of reducing agent could promote the release of LOR-2501. In the in vitro cell evaluation experiment, the results of MTT anti tumor experiment and flow cytometry assay showed that the prepared PEI-SS-OA vector could transfect LOR-2501 into He La and A549 cells and inhibit the growth of tumor cells. The laser confocal microscope observed that the PEI-SS-OA/LOR-2501 complex could enter into large quantities. The results of R1 m RNA measured by real time fluorescence quantitative PCR showed that the decrease efficiency of R1 m RNA in He La and A549 cells was 40.8% and 43.6% respectively. On the basis of PEI, a polymer containing amide bond (N-H) was synthesized on the basis of PEI. The hydrophobic modification (named PEI-NH-OA) was carried out with oil acyl (OA). The PEI-NH-OA drug delivery system.PEI-NH-OA was prepared by ethanol injection. The optimal preparation conditions were obtained. The particle size is 102 + 3.9 nm, the zeta potential value is 54.5 + 3.1 m V.PEI-NH-OA and the toxicity of He La and A549 cells is more than 90%. In the 30 days, the particle size and zeta potential of the PEI-NH-OA loading system are not obviously changed. The particle size of the PEI-NH-OA drug system is smaller and the cation characteristics are more than that of the PEI-SS-OA drug system. The establishment and evaluation of.4.PEI-NH-OA/LOR-2501 transmission system with better stability was established and the PEI-NH-OA/LOR-2501 transfer system was established at the same time, and the transfection efficiency of PEI-NH-OA was evaluated. The optimum N/P was 6:1. at the time of N/P as the index of particle size and zeta potential, and PEI-NH-OA and LOR-2501 have been fully combined to form nanocomposites. The antitumor experiment showed that the inhibitory rate of the complex on He La and A549 cells was 46.2% and 41.5%., respectively, by flow cytometry and laser confocal microscopy, and found that the complex was able to enter into He La and A549 tumor cells in large quantities. The down regulation efficiency of R1 m RNA in the cells was 37.7% and 34.5% respectively, while the PEI group was only 20%.Western-blot to determine R1 protein results, and the down regulation of R1 protein was 50.1% and 46.3%. visible PEI-NH-OA could efficiently transfect LOR-2501 into cells, which was more stable and smaller than PEI-SS-OA, and selected in the later experiment. On the basis of PEI-NH-OA/LOR-2501 complex, this paper further modifies the.5.FA/PEI-NH-OA/LOR-2501 transfer system and further combines the Folic acid (FA) with the above complex by electrostatic adsorption to obtain the FA/PEI-NH-OA/LOR-2501 transfer system.FA/PEI-NH-OA/LOR-2501 complex at the optimum proportion. The diameter was 159 + 1.1 nm and the value of zeta potential was -12.2 + 1.3 m V. The PEI-NH-OA carrier after adding FA was still able to combine with LOR-2501 without obvious toxicity, and the cell viability was above 80%. The inhibitory rate of He La and A549 cells was 55.6% and the flow cytometry was detected in the anti tumor evaluation experiment of the cells in vitro. The results showed that the average fluorescence intensity of the complex in He La cells with high expression of folate receptor (FR+) and the low expression of folate receptor (FR-) in A549 cells was more than 2 times more than before FA modification, and the same phenomenon appeared in KB cells (FR+) and SK-HEP-1 cells (FR-). The results of laser confocal microscopy showed that the complex could be a large amount. The results of.R1 m RNA assay in the tumor cells showed that in He La and A549 cells, the RNA downregulation efficiency of R1 m RNA was 51.7% and 45.7%.Western-blot showed R1 protein, and the downregulation efficiency of R1 protein was 72% and 68.9% respectively. The effect of free FA on the entry of FA/PEI-NH-OA/LOR-2501 nanocomposites into tumor cells was first investigated. The results showed that the free FA group with a concentration of 0.01,0.1,1 mmol/L was compared with that of the free FA group, no matter in He La, KB cell (FR+) or A549, and there was no one in the uptake of the complex. The effect of three endocytic pathway inhibitors, sucrose, cytosin D and nystatin on endocytosis, is also investigated. The results show that the complex may be mainly through the endocytic pathway mediated by the gridin. The laser copolymerization is used after three kinds of internalized pathway specific dye labeling cells. The results further verify that the complex is mainly through the endocytic pathway mediated by the gridin to enter the tumor cells. To sum up, the hydrophobic modified PEI and its FA modified carrier system have low toxicity and high efficiency, and the transfection efficiency of the FA/PEI-NH-OA drug delivery system The expression level of FR on tumor cells is independent, and has high transfection efficiency in a variety of tumor cell lines. It is expected to become a safe and efficient AS-ODN drug carrier.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R943

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