Naumann Christoph A, Prucker O, Lehmann T, Rühe J, Knoll W, Frank C W
Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
Biomacromolecules. 2002 Jan-Feb;3(1):27-35. doi: 10.1021/bm0100211.
We present a new molecular engineering approach in which a polymer-supported phospholipid bilayer is vertically stabilized by controlled covalent tethering at both the polymer-substrate and polymer-bilayer interfaces. This approach is based on lipopolymer molecules, which not only form a polymer cushion between the phospholipid bilayer and a solid glass substrate but also act as covalent connections (tethers) between the bilayer and cushion. Our approach involves Langmuir-Blodgett transfer of a phospholipid-lipopolymer monolayer followed by Schaefer transfer of a pure phospholipid monolayer and is capable of varying the tethering density between the polymer layer and the phospholipid bilayer in a very controlled manner. Further stabilization is achieved if the glass substrate is surface-functionalized with a benzophenone silane. In this case, a photocross-linking reaction between the polymer and benzophenone group allows for the covalent attachment of the polymer cushion to the glass substrate. This approach is similar to that recently reported by Wagner and Tamm in which double tethering is achieved via lipopolymer silanes (Wagner, M. L.; Tamm, L. K. Biophys. J. 2000, 79, 1400). To obtain a deeper understanding of how the covalent tethering affects the lateral mobility of the bilayer, we performed fluorescence recovery after photobleaching (FRAP) experiments on polymer-tethered bilayers at different tethering densities (lipopolymer/phospholipid molar ratios). The FRAP data clearly indicate that the hydrophobic lipopolymer moieties act as rather immobile obstacles within the phospholipid bilayer, thereby leading to hindered diffusion of phospholipids. Whereas the high lateral diffusion coefficient of D = 17.7 mum(2)/s measured at low tethering density (5 mol % lipopolymer) indicates rather unrestricted motion within the bilayer, corresponding values at moderate (10 mol % lipopolymer) and high (30 mol % lipopolymer) tethering densities of D = 9.7 mum(2)/s and D = 1.1 mum(2)/s, respectively, show significant hindered diffusion. These results are contrary to the recent findings on similar membrane systems reported by Wagner and Tamm in which no significant change in phospholipid diffusion was found between 0 and 10 mol % lipopolymer. Our experimental report leads to a deeper understanding of the complex problem of interlayer coupling and offers a path toward a compromise between stability of the whole system and lateral mobility within the bilayer. Furthermore, the FRAP measurements show that polymer-tethered membranes are very interesting model systems for studying problems of restricted diffusion within two-dimensional fluids.
我们提出了一种新的分子工程方法，其中通过在聚合物 - 底物和聚合物 - 双层界面处进行可控的共价连接来垂直稳定聚合物支撑的磷脂双层。该方法基于脂聚合物分子，其不仅在磷脂双层和固体玻璃底物之间形成聚合物垫层，而且还作为双层和垫层之间的共价连接（系链）。我们的方法包括磷脂 - 脂聚合物单层的朗缪尔 - 布洛杰特转移，然后是纯磷脂单层的谢弗转移，并且能够以非常可控的方式改变聚合物层和磷脂双层之间的连接密度。如果玻璃底物用二苯甲酮硅烷进行表面功能化，则可实现进一步的稳定化。在这种情况下，聚合物和二苯甲酮基团之间的光交联反应允许聚合物垫层与玻璃底物共价连接。该方法类似于瓦格纳和塔姆最近报道的方法，其中通过脂聚合物硅烷实现双连接（瓦格纳，M. L.；塔姆，L. K.《生物物理学杂志》2000年，79卷，1400页）。为了更深入地了解共价连接如何影响双层的横向流动性，我们对不同连接密度（脂聚合物/磷脂摩尔比）的聚合物连接双层进行了光漂白后荧光恢复（FRAP）实验。FRAP数据清楚地表明，疏水性脂聚合物部分在磷脂双层中充当相当固定的障碍物，从而导致磷脂扩散受阻。在低连接密度（5摩尔％脂聚合物）下测得的高横向扩散系数D = 17.7μm²/s表明双层内的运动相当不受限制，而在中等（10摩尔％脂聚合物）和高（30摩尔％脂聚合物）连接密度下的相应值分别为D = 9.7μm²/s和D = 1.1μm²/s，显示出明显的扩散受阻。这些结果与瓦格纳和塔姆最近关于类似膜系统的发现相反，在他们的研究中，在0至10摩尔％脂聚合物之间未发现磷脂扩散有显著变化。我们的实验报告有助于更深入地理解层间耦合的复杂问题，并为在整个系统的稳定性和双层内的横向流动性之间达成折衷提供了一条途径。此外，FRAP测量表明，聚合物连接的膜是研究二维流体中受限扩散问题的非常有趣的模型系统。
