【作者】 姜嫣嫣；

【导师】 裴元英；

【作者基本信息】 复旦大学 ， 药剂学， 2005， 博士

【摘要】 人体内存在于细胞膜或胞内蛋白上的受体可特异性地识别配体分子,并将其导入到体内特定的位置。由于配体无毒、无免疫原性、可生物降解,可利用配体与药物形成复合物将药物特异性地导入体内某一部位。因此,配体-受体介导的主动靶向给药系统备受关注。转铁蛋白(Tf)是一种参与机体铁离子运输的糖蛋白。转铁蛋白和转铁蛋白受体介导的内吞作用是生物细胞最具特点的转运过程之一。由于转铁蛋白受体在肿瘤细胞和快速增殖的细胞表面高度表达,已有研究将转铁蛋白作为配体一受体介导药物传递系统的导向基团来治疗癌症。 蛋白质和多肽药物的聚乙二醇(PEG)化可使药物半衰期延长,促进药物被动靶向肿瘤组织。重组人肿瘤坏死因子(rHuTNF-α)是一抗肿瘤细胞因子,对各种肿瘤细胞有直接细胞毒作用,可激活机体的抗肿瘤免疫反映,可引起肿瘤的缺血性坏死。若希望通过静脉给药获得明显的抗肿瘤效果,则必须持续大剂量注射,进而导致严重的毒副作用。PEG化rHuTNF-α可延长药物的半衰期,增加肿瘤组织药物的蓄积和提高抗肿瘤活性。 为进一步提高PEG-rHuTNF-α的肿瘤靶向作用,减小给药剂量,改善治疗效果,本研究以具有双功能基团的NHS-PEG-MAL为“桥梁”,将Tf与PEG化rHuTNF-α结合,形成Tf-PEG-rHuTNF-α复合物。这种复合物具有主动靶向和聚乙二醇被动靶向的双重功能。 由于rHuTNF-α价格昂贵,本研究首先选择β-乳球蛋白B(LG)为模型蛋白,探讨将Tf与蛋白质连接的可能性以及靶向肿瘤细胞的效果。在Tf-PEG-LG的合成获得成功的基础上,我们成功地将PEG化的rHuTNF-α与Tf进行共价连接,获得具有主动和被动靶向肿瘤细胞双重功能的Tf-PEG-rHuTNF-α复合物。 合成反应的第一步是PEG化LG或PEG化rHuTNF-α,然后通过PEG一端的马来酰亚胺基团(MAL)将MAL-PEG-LG或MAL-PEG-rHuTNF-α与巯基化的Tf连接形成Tf-PEG-LG或Tf-PEG-rHuTNF-α。整个反应过程均在室温或4℃下,中性缓冲液中进行,反应条件温和,适合不稳定蛋白质类药物的修饰。 LG和rHuTNF-α分别被不同摩尔比的PEG修饰。不同反应时间下,进行SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)测定。采用Image Master图象分析系统定量凝胶中游离蛋白质,评价蛋白质的PEG化程度;结果显示SDS-PAGE法可使游离蛋白质与PEG化蛋白质达到完全分离。蛋白质的PEG化程度随PEG量和反应时间的增加而提高。更多还原

【Abstract】 Ligand-receptor-mediated delivery systems have received major attention in the past few years because naturally occurring ligands and their receptors are not only able to achieve site-specific targeting to ligand-specific biosites, but they are also biodegradable, non-toxic and non-immunogenic. Transferrin (Tf) is a glycoprotein which transports a ferric ion in the body. A major pathway for cellular iron uptake is by internalization of the complex of iron-bound transferrin and the transferring receptor (TfR). Transferrin and TfR-mediated endocytosis is one of the best-characterized processes in cell biology. It was shown earlier that the number of TfR on the cell surface is increased significantly in tumor cells and other rapidly proliferating cells compared with slowly growing non-malignant cell populations. Tf is a stable, commercially available protein and has been used as a ligand-receptor-mediated delivery system for cancer chemotherapy.The conjugation of polyethylene glycol (PEG), PEGylation, is a procedure of growing interest for enhancing the therapeutic and biotechnological potential of peptides and proteins, and has been extensively used to modify various proteins. PEGylation has been considered a tool to enhance the drug delivery to neoplastic tissues by the passive enhanced permeation and retention mechanism.Recombinant human tumor necrosis factor alpha (rHuTNF-α, Mr= 17000), an anti-tumor cytokine, exhibits striking biological effects, such as direct cytotoxicity against various kinds of tumor cells, activation of immune anti-tumor response, and inducement of hemorrhagic necrosis of certain transplanted solid tumors. However, excessively frequent and high dose of rHuTNF-a are required for significant anti-tumor effects because of its short plasma half-life and it was found to have severe toxic side-effects. We have previously demonstrated that PEGylated rHuTNF-a had a longer plasma half-life, higher level accumulation in tumor tissues and better anti-tumor potency than unmodified one.The main goal for this study is to synthesize Tf-PEG- rHuTNF-a conjugates by binding Tf to PEG-rHuTNF-a, using heterofunctional NHS-PEG-MAL as a spacer. The Tf-PEG-rHuTNF-α conjugates may both maintain the advantages of PEGylation and achieve the function of active targeting tumor cells.Because rHuTNF-a is very expensive, in this study, P-lactoglobulin B (Mr= 18276, LG) was firstly selected as a model protein in order to explored the possibility of coupling Tf to PEGylation protein. On the basis of the successful synthesis of the Tf-PEG-LG conjugate, the Tf-PEG-rHuTNF-a conjugate was also prepared.The first step of synthesis was to prepare MAL-PEG-LG (or MAL-PEG-rHuTNF-a) via the formation of an amino bond between lysine amino groups of protein and the terminal N-hydroxylsuccinimide (NHS) group of NHS-PEG-MAL. Subsequently, the Tf-PEG-LG conjugates was synthesize by reacting the double bond of the maleimide (MAL) group at the other end terminus of MAL-PEG-LG (or MAL-PEG-rHuTNF-a) with thiolated human transferrin forming a stable thioether bond.LG and rHuTNF-awere modified with different molar ratio of NHS-PEG-MAL and sampled at different reaction time. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was applied to determine the resulted PEGylated protein which the free protein and PEG-protein were completely separated by SDS-PAGE. The free protein band in SDS-gel was quantified by using the ImageMaster bio-imaging system, and then the degree of PEGylation were assessed. The degree of PEGylated LG and rHuTNF-a increased with increasing mass of PEG and reaction time.MAL-PEG-LG were separated and purified by gel filtration chromatography. The yield of purified MAL-PEG- LG was 51.6%, and the molar ratio of LG and PEG was 1:7.4. The Tf-PEG-LG conjugate was prepared by conjugating MAL-PEG-LG to thiolated Tf. After isolated by gel filtration chromatography, the Tf-PEG-LG conjugate was obtained at a molar ratio of 1:1.4 (LG/Tf). The total yield (base on LG) was 28.2%.MAL-PEG-rHuTNF-a was purified from the reation mixture by using cation-exchange perfusion chromatography and the yield was 85.7%. The molar ratio of rHuTNF-a and PEG was 1:4.8. The synthesis of Tf-PEG-rHuTNF-a conjugate was achieved at a molar ratio of 1:1.1 (rHuTNF-a/Tf). The total yield (based on rHuTNF-a) was 45.2%.To assess the specificity of the Tf conjugates binding, TfR expression was determined in K562, KB, S-l 80 and L02 cells by determining their maximum binding sites and affinity using the classic radioligand binding analysis. The results showed that K562, KB and S-l80 tumor cells expressed more TfR than human normal更多还原