Design of Cell-Surface-Retained Polymers for Artificial Ligand Display
Design of Cell-Surface-Retained Polymers for Artificial Ligand Display
复制标题
DOI：
10.1002/cbic.200800621
复制
发表时间：
2009-01-26
期刊：
影响因子：
3.2
通讯作者：
Ijiro, Kuniharu
中科院分区：
文献类型：
--
作者：
Kamitani, Ryosuke;Niikura, Kenichi;Ijiro, Kuniharu
细胞表面工程仍然是生物技术和医学科学的一个重要领域，因为这些技术允许将定制的系统添加到细胞中，从而能够通过人工信号修改或调节细胞行为哺乳动物细胞表面工程的几种方法已被探索。例如，低聚糖前体的代谢结合允许随后的共价附着在所需要的合成表位到细胞表面最近，一些研究小组报道了利用脂系分子在细胞表面被动插入合成分子的非共价显示。[3,4]这些方法将适用于广泛的细胞类型，并且它们对固有信号转导的干扰较小。Peterson和他的同事们已经证明，非天然的膜锚，n-烷基-3β-胆固醇胺，当与蛋白质和药物的结合基序连接时，作为一种有效的药物传递的人工受体添加的配体通过受体介导的内切胞浆作用迅速传递到细胞中。Nagamune和他的同事们也报道了使用PEG-脂质，在PEG链的末端附着一个单一的功能基团来锚定蛋白质我们的目标是创造能够长时间保留在细胞表面的分子，并作为细胞表面显示人工识别位点的有效支架。在本文中，我们报告了我们的发现，在聚合物主链上添加二级氨基可以增加在细胞膜上的保留时间，而不会快速内化（图1）。在含仲胺聚合物的末端引入脂质片段，增强了聚合物在细胞表面的结合，但不影响其在细胞中的定位。共聚焦激光扫描（CLS）显微镜和流式细胞术分析显示，含仲胺的聚合物在细胞表面保留超过12小时，而含原胺的聚合物则迅速内化。这些含仲氨基的聚合物可以作为人工配体在细胞膜上展示的有效支架。显示在HeLa细胞表面的配体可以有效地参与表面结合，从而为通过对这些配体的特异性识别来控制细胞粘附事件提供了一种新的技术。以油基引发剂1 （Scheme1）为原料，采用原子转移自由基聚合（ATRP）法制备了含环氧化合物的脂系聚合物3 （Mn: 12520, Mw: 17555，多分散性指数：1.40）。脂质部分拴在聚甲基丙烯酸缩水甘油酯（PGMA）上；PGMA引起了人们极大的兴趣，因为用环氧基团对其进行进一步改性将为在聚合物链中引入多种官能团提供一种简单的合成途径油基链被用作膜锚，因为它不影响细胞信号传导油基- pgma 3与FITC偶联，随后与乙醇胺、铵或硫酸反应，分别得到标记聚合物5、6和7。在与乙醇胺偶联后，聚合物带有正电荷（由于二级氨基）并且是水溶性的通过在氨溶液中的反应，伯胺基被引入到聚合物侧链上酸催化的环氧化合物开孔反应提供了一种水溶性非离子聚合物阳离子聚合物5、6和非离子聚合物7分别与HeLa细胞孵育10 min，用CLS法观察聚合物的定位。
Cell surface engineering remains an important field in biotechnology and medical science, as these techniques allow the ACHTUNGTRENNUNGaddition of tailored systems into cells and thus enable the modification or regulation of cellular behavior through artificial signals.[1] Several approaches to the engineering of the mammalian cell surface have been explored. For example, metabolic incorporation of oligosaccharide precursors allows for subsequent covalent attachment of desired synthetic epitopes onto the cell surface.[2] Recently, several groups have reported the noncovalent display of synthetic molecules on the cellular surface by passive insertion using lipid-tethered molecules.[3, 4] These methods will be applicable for wide range of cell types, and they cause less perturbation of inherent signal transduction. Peterson and co-workers have shown that the non-natural membrane anchor, N-alkyl-3β-cholesterylamine, acts as an artificial receptor for efficient drug delivery when linked to the binding motifs for proteins and drugs.[3] Added ligands are rapidly delivered into the cell through receptor-mediated endo-ACHTUNGTRENNUNGcytosis. Nagamune and co-workers have also reported the use of PEG-lipids with a single functional group attached on the termini of the PEG chain to anchor proteins.[4] Our aim is to create molecules that can be retained on the cell surface for a long period and function as an effective scaffold for the display of artificial recognition sites on the cellular surface. In this paper, we report our finding that the addition of secondary amino groups to the polymer backbone affords increased retention time on the cell membrane without rapid internalization (Figure 1). Introduction of a lipid moiety to the end of the secondary amine-containing polymer enhanced binding of the polymer on the cell surface but did not affect localization in the cells. Confocal laser scanning (CLS) microscopy and flow cytometry analysis revealed that the secondary amine-containing polymers were retained on the cell surface for over 12 h. In contrast, primary amine-containing polymers were rapidly internalized. These secondary amino group-containing polymers can act as an effective scaffold for the display of artificial ligands on the cell membrane. Ligands displayed on the surface of HeLa cells can efficiently participate in surface binding, thus affording a new technique for the control of cell adhesion events through the specific recognition of these ligands.We synthesized the epoxide-bearing, lipid-tethered polymer 3 (Mn: 12520, Mw: 17555, polydispersity index: 1.40) by atom transfer radical polymerization (ATRP) with oleyl initiator 1 (Scheme1). The lipid moiety was tethered to poly (glycidyl methacrylate)(PGMA); PGMA is of great interest, as its further modification with epoxide groups would offer a simple synthetic route to the introduction of a variety of functional groups into a polymer chain.[5] The oleyl chain was employed as a membrane anchor because it did not affect cell signalling.[4] Oleyl-PGMA 3 was conjugated with FITC and subsequently reacted with ethanolamine, ammonium or sulfuric acid to provide labeled polymers 5, 6 and 7, respectively. After coupling with ethanolamine, the polymer has a positive charge (due to secondary amino groups) and is water soluble.[6] The primary amino groups were introduced to the polymer side chain through a reaction in ammonia solution.[7] The acid-catalyzed epoxide-opening reaction provides a water-soluble nonionic polymer.[8] Cationic polymers 5 and 6 and nonionic polymer 7 were incubated separately with HeLa cells for 10 min, and the localization of the polymer was observed by using CLS …