Tumor cell targeting of transferrin-PEG-TNF-α conjugate via a receptor-mediated delivery system:: Design, synthesis, and biological evaluation
Tumor cell targeting of transferrin-PEG-TNF-α conjugate via a receptor-mediated delivery system:: Design, synthesis, and biological evaluation
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DOI：
10.1021/bc060135f
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发表时间：
2007-01-01
影响因子：
4.7
通讯作者：
Pei, Yuan-Ying
中科院分区：
文献类型：
--
作者：
Jiang, Yan-Yan;Liu, Chen;Pei, Yuan-Ying
聚乙二醇化是一个越来越感兴趣的程序，用于增强肽和蛋白质的治疗和生物技术潜力。转铁蛋白（Tf）已被提出可用于靶向癌细胞。本研究旨在用转铁蛋白（Tf）修饰聚乙二醇化重组人肿瘤坏死因子α（PEG-TNF-α），形成Tf-PEG-TNF-α偶联物，既保持聚乙二醇化的优点，又实现主动靶向肿瘤细胞的功能。在用5倍、20倍、40倍和60倍摩尔过量的3.4 kDa N-羟基琥珀酰亚胺-PEG-马来酰亚胺（分别为PT 1、PT 2、PT 3和PT 4）进行的PEG化反应中，合成了具有不同PEG链的PEG-TNF-α缀合物。引入使用阳离子交换柱的灌注层析技术来纯化PEG-TNF-α结合物。具有约五个PEG链的PT 4由于最高程度的PEG化和最大反应产率而被选择为主要候选物。巯基化的Tf与PEG-TNF-α缀合物上的PEG链远端的马来酰亚胺基团缀合，纯化后所得的Tf-PEG-TNF-alpha缀合物在一个TNF-alpha分子上含有大约一个Tf配体。以转铁蛋白和酪氨酸蛋白4（TPT 4）为偶联物，研究了TPT 4对K562和KB两种肿瘤细胞上转铁蛋白受体（TfR）的特异性和亲和力。使用放射性配体结合分析进行受体结合测定和竞争实验。结果表明，TPT 4和Tf特异性结合肿瘤细胞表面的TfR，并且缀合物对TfR的亲和力与天然Tf相似。相比之下，PEG-TNF-α没有特异性。在S-180荷瘤小鼠体内进行了生物分布和抗肿瘤作用的研究。发现TPT 4可显著改变TNF-α的体内行为特征。与TNF-α和PT 4相比，肿瘤组织中外渗的TPT 4表现出显著延迟的血液清除和最高的肿瘤内TNF-α水平。此外，TPT 4的肿瘤抑制率比TNF-α和PT 4的肿瘤抑制率提高了5.3倍和1.8倍，表明TPT 4表现出最高的抗肿瘤活性。这些结果表明，Tf-PEG-TNF-α是一种有用的长循环缀合物，具有特异性受体结合的能力，从而增强TNF-α的抗肿瘤活性。
PEGylation is a procedure of growing interest for enhancing the therapeutic and biotechnological potential of peptides and proteins. Transferrin (Tf) has been proposed to be useful for targeting cancer cells. The aim of this study was to modify PEGylated recombinant human tumor necrosis factor alpha (PEG-TNF-alpha) with Tf to form Tf-PEG-TNF-alpha conjugates, which would maintain the advantages of PEGylation and also achieve the function of active targeting to tumor cells. In PEGylation reactions with 5-, 20-, 40-, and 60-fold molar excess of 3.4 kDa N-hydroxysuccinimide-PEG-maleimide (PT1, PT2, PT3, and PT4, respectively), PEG-TNF-alpha conjugates with different PEG chains were synthesized. A perfusion chromatography technique using a cation-exchange column was introduced to purify PEG-TNF-alpha conjugates. PT4 with about five PEG chains was selected as a lead candidate due to highest extent of PEGylation and maximum reaction yield. Thiolated Tf was conjugated to the maleimide group at the distal end of the PEG chains on the PEG-TNF-alpha conjugates, with the resulting Tf-PEG-TNF-alpha conjugates after purification containing approximately one Tf ligand on one TNF-alpha molecule. The conjugate of Tf and PT4 (TPT4) was selected to assess the specificity and affinity to transferrin receptor (TfR) on two kinds of tumor cells, K562 and KB. Both the receptor binding assays and the competition experiments were performed using radioligand binding analysis. The results demonstrated that TPT4 as well as Tf bound specifically to the TfR on the tumor cell surface and the affinity of the conjugate to TfR was similar to that of native Tf. In contrast, PEG-TNF-alpha demonstrated no specificity. The biodistribution and antitumor effects were investigated in S-180 tumor-bearing mice. It was found that TPT4 could markedly alter in vivo behavioral characteristics of TNF-alpha. Compared with TNF-alpha and PT4, extravasated TPT4 in tumor tissues exhibited a significantly delayed blood clearance and the highest intratumoral TNF-alpha levels. Furthermore, the inhibitory rate of tumor of TPT4 enhanced 5.3- and 1.8-fold over that of TNF-alpha and PT4, indicating that TPT4 exhibited the highest antitumor activity. These results suggested that Tf-PEG-TNF-alpha was a useful long circulating conjugate with the capabilities of specific receptor binding resulting in enhanced antitumor activity of TNF-alpha.