Castelletto Valeria, de Mello Lucas R, Seitsonen Jani, Hamley Ian W
School of Chemistry, Food Biosciences and Pharmacy, University of Reading, Whiteknights, Reading RG6 6AD, U.K.
Nanomicroscopy Center, Aalto University, Puumiehenkuja 2, FIN-02150 Espoo, Finland.
ACS Appl Mater Interfaces. 2024 Dec 18;16(50):68713-68723. doi: 10.1021/acsami.4c18165. Epub 2024 Dec 9.
Short bioactive peptide sequences are of great interest in biomaterials development. We investigate the self-assembly of a lipopeptide containing both the highly cationic CSK toll-like receptor agonist hexapeptide sequence and RGDS integrin-binding motif, i.e., C-CSKRGDS, as well as the control containing a scrambled terminal sequence C-CSKGRDS. Both lipopeptides are found to form micelles, as revealed by small-angle X-ray scattering and cryogenic transmission electron microscopy, and modeled using atomistic molecular dynamics simulations. We carefully examined methods to probe the aggregation of the molecules, i.e. to obtain the critical micelle concentration (CMC). Fluorescent probe assays using 1-anilino-8-naphthalenesulfonate (ANS) reveal low CMC values, 1-2 μM, which contrast with consistent values more than 2 orders of magnitude larger obtained from surface tension and electrical conductivity as well as unexpected UV/vis absorption spectra discontinuities and fluoresccence probe assays using Nile red. The anomalous results obtained from an ANS fluorescence probe are ascribed to the effect of ANS binding to the cationic (lysine and arginine) residues in the lipopeptide, which leads to a conformational change, as shown by circular dichroism, even at low concentrations below the actual CMC. Despite the small change in the peptide sequence (swapping of G and R residues), there is surprisingly a significant difference in the aggregation propensity and association number, both of which are greater for C-CSKGRDS. Both lipopeptides are cytocompatible (with fibroblasts and myoblasts) at low concentration, although cytotoxicity is noted at higher concentration.
短生物活性肽序列在生物材料开发中备受关注。我们研究了一种脂肽的自组装情况，该脂肽同时包含高度阳离子化的CSK Toll样受体激动剂六肽序列和RGDS整合素结合基序，即C-CSKRGDS，以及含有乱序末端序列C-CSKGRDS的对照物。小角X射线散射和低温透射电子显微镜显示，这两种脂肽均能形成胶束，并通过原子分子动力学模拟进行建模。我们仔细研究了探测分子聚集的方法，即获得临界胶束浓度（CMC）。使用1-苯胺基-8-萘磺酸盐（ANS）的荧光探针测定法显示CMC值较低，为1-2μM，这与通过表面张力、电导率获得的比其大2个数量级以上的一致值形成对比，同时也与使用尼罗红的紫外/可见吸收光谱不连续性和荧光探针测定法的意外结果形成对比。ANS荧光探针得到的异常结果归因于ANS与脂肽中阳离子（赖氨酸和精氨酸）残基结合的效应，这会导致构象变化，圆二色性表明即使在低于实际CMC的低浓度下也是如此。尽管肽序列变化很小（G和R残基的交换），但令人惊讶的是，聚集倾向和缔合数存在显著差异，两者对于C-CSKGRDS都更大。两种脂肽在低浓度下均具有细胞相容性（对成纤维细胞和平滑肌细胞），尽管在较高浓度下会观察到细胞毒性。
