Using "click" chemistry to prepare SAM substrates to study stem cell adhesion.
Using "click" chemistry to prepare SAM substrates to study stem cell adhesion.
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DOI：
10.1021/la804077t
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发表时间：
2009-05-19
期刊：
影响因子：
--
通讯作者：
Murphy WL
中科院分区：
文献类型：
--
作者：
Hudalla GA;Murphy WL
我们利用铜(I)催化的叠氮-炔环加成(CuAAC)“点击”反应制备了细胞黏附肽Arg-Gly-Asp-Ser-Pro(RGDSP)的自组装单分子膜(SAM)。表面制备方法包括首先用叠氮封端的乙二醇烷硫酸酯(HS-EG6-N3)和三甘醇硫代烷基硫酸酯(HS-EG3)形成混合自组装膜，然后利用CuAAC反应固定端炔基肽。混合自组装膜被归类为生物惰性自组装膜，由10摩尔%HS-EG6-N3和90摩尔%HS-EG3组成的自组装膜在1 mg/ml溶菌酶或10%胎牛血清溶液中表现出极小的非特异性蛋白质吸附。在铜(I)-tBTA络合物存在下，乙炔封端的多肽与叠氮封端的自组装膜之间的反应快速而定量地进行，反应符合准一级动力学，反应速率常数为∼0.2min−1。在自组装膜形成过程中，改变HS-EG6-N3与HS-EG3的比例可以控制叠氮的密度，进而控制底物上的RGDSP的密度。因此，这些底物被用来研究RGDSP表面密度与人骨髓间充质干细胞(HMSC)黏附、铺展和局部黏附复合体形成的详细关系，而不受非特异性吸附血清蛋白的干扰。结果表明，RGDSP36 nm或更小的分子间距(≥表面的摩尔百分数为0.01%)对hMSC的黏附是足够的，而11 nm或更小的间距(≥在表面的摩尔百分比为0.05时)足以使细胞铺展和形成焦点黏附复合体。总而言之，我们的结果表明，CuAAC是多肽与其他生物惰性自组装膜结合的合适机制，所得到的自组装膜可用于研究多肽密度对干细胞行为的依赖。
We have used a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) “click” reaction to prepare self-assembled monolayers (SAMs) presenting the cell adhesion peptide Arg-Gly-Asp-Ser-Pro (RGDSP) in a bio-inert background. The surface preparation approach involved first forming mixed SAMs with an azide-terminated hexaethylene glycol alkanethiolate (HS---EG6---N3) and a triethylene glycol alkanethiolate (HS---EG3), then using the CuAAC reaction to immobilize an alkyne-terminated peptide. The mixed SAMs were classified as bio-inert, as SAMs comprised of 10 mole percent HS---EG6---N3 and 90 mole percent HS---EG3 showed minimal non-specific protein adsorption in solutions of 1 mg/ml lysozyme or 10% fetal bovine serum. The reaction between an acetylene-terminated peptide and an azide-terminated SAM proceeded rapidly and quantitatively in the presence of a Cu(I)-TBTA complex, displaying pseudo-first order kinetics with a rate constant of ∼ 0.2 min−1. Varying the ratio of HS---EG6---N3 to HS---EG3 during SAM formation allowed for control over the density of azide and, in turn, the density of RGDSP on the substrates. These substrates were therefore used to study the detailed relationship between RGDSP surface density and human mesenchymal stem cell (hMSC) adhesion, spreading, and focal adhesion complex formation, without interference from non-specifically adsorbed serum proteins. Results indicate that an RGDSP intermolecular spacing of 36 nm or less (≥ 0.01 mole percent on the surface) is sufficient for hMSC adhesion and a spacing of 11 nm or less (≥ 0.05 mole percent on the surface) is sufficient for cell spreading and focal adhesion complex formation. In total, our results demonstrate that CuAAC is a suitable mechanism for conjugating peptides to otherwise bio-inert SAMs, and that the resulting SAMs can be used to study the dependence of peptide density on stem cell behavior.
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