Castelletto Valeria, de Mello Lucas R, Pelin Juliane, Hamley Ian W
School of Chemistry, Food Biosciences and Pharmacy, University of Reading, Whiteknights, Reading RG6 6AD, U.K.
Currently at Departamento de Ciências Farmacêuticas, Universidade Federal de São Paulo, Diadema, São Paulo 09913-030, Brazil.
Bioconjug Chem. 2025 Apr 16;36(4):792-802. doi: 10.1021/acs.bioconjchem.5c00051. Epub 2025 Apr 2.
Toll-like receptor (TLR) agonists are of interest in immunotherapy and cancer vaccines. The most common agonists of TLR2 are based on PamCys or PamCys. In the former, two palmitoyl (Pam) fatty acids are linked to a glycerylcysteine motif by ester linkages. PamCys is analogous but contains an extra Pam group on the α-amine. Here, we compare the self-assembly in aqueous solution of the parent PamCysOH and PamCys amino acid conjugates to that of PamCysSK and PamCysSK which are potent TLR2 agonists bearing the CysSK peptide sequence. All four conjugates exhibit a critical aggregation concentration above which self-assembled structures are formed. We find through a combination of small-angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM), and confocal fluorescence microscopy remarkable differences in self-assembled nanostructures. PamCysOH and PamCysOH both form unilamellar vesicles, although these are larger for the latter compound, an effect ascribed to enhanced membrane rigidity. This is in contrast to previously reported morphologies for PamCysSK and PamCysSK4, which are spherical micelles or predominantly wormlike micelles, respectively [Hamley, I. W.; et al. . Chem. Comm. 2014, 50, 15948-15951]. We also examine the effect of introduction in the bulky -terminal Fmoc [fluorenylmethoxycarbonyl] group on the self-assembly of Fmoc-PamCysOH. This compound also forms vesicles (above a critical aggregation concentration, determined from dye probe fluorescence experiments) in aqueous solution, larger than those for PamCysOH and with a population of perforated/compound vesicles. The carboxyl-coated (and amino-coated for PamCysOH) vesicles demonstrated here represent a promising system for future development toward bionanotechnology applications such as immune therapies. Conjugates PamCysOH, PamCysSK, and PamCysSK show good cytocompatibility at low concentrations, and in fact, the cell compatibility extends over a wider concentration range for PamCysOH. The TLR2/6 agonist activity was assessed using an assay that probes secreted alkaline phosphatase (SEAP) in NF-κB-SEAP reporter HEK293 cells expressing human TLR2 and TLR6, and PamCySOH shows significant activity, although not to the extent of PamCysSK4 or PamCysSK. Thus, PamCysOH in particular is of interest as a vesicle-forming TLR2/6 agonist and stimulator of immune response.
Toll样受体（TLR）激动剂在免疫治疗和癌症疫苗领域备受关注。TLR2最常见的激动剂基于PamCys或PamCys。在前者中，两个棕榈酰（Pam）脂肪酸通过酯键与甘油半胱氨酸基序相连。PamCys类似，但在α-氨基上含有一个额外的Pam基团。在此，我们比较了母体PamCysOH和PamCys氨基酸缀合物在水溶液中的自组装情况与PamCysSK和PamCysSK的自组装情况，PamCysSK和PamCysSK是带有CysSK肽序列的强效TLR2激动剂。所有四种缀合物都表现出一个临界聚集浓度，高于此浓度会形成自组装结构。我们通过小角X射线散射（SAXS）、低温透射电子显微镜（cryo-TEM）和共聚焦荧光显微镜相结合的方法，发现自组装纳米结构存在显著差异。PamCysOH和PamCysOH都形成单层囊泡，尽管后者的囊泡更大，这一效应归因于膜刚性增强。这与之前报道的PamCysSK和PamCysSK4的形态形成对比，它们分别是球形胶束或主要是蠕虫状胶束[Hamley, I. W.; 等人。《化学通讯》。2014年，50卷，15948 - 15951页]。我们还研究了在庞大的末端芴甲氧羰基（Fmoc）基团引入对Fmoc - PamCysOH自组装的影响。该化合物在水溶液中也形成囊泡（高于由染料探针荧光实验确定的临界聚集浓度），比PamCysOH形成的囊泡更大，并且有一群穿孔/复合囊泡。这里展示的羧基包被（对于PamCysOH是氨基包被）的囊泡代表了一个有前途的系统，有望在未来朝着生物纳米技术应用（如免疫疗法）发展。缀合物PamCysOH、PamCysSK和PamCysSK在低浓度下表现出良好的细胞相容性，事实上，PamCysOH的细胞相容性在更宽的浓度范围内都存在。使用一种检测方法评估TLR2/6激动剂活性，该方法检测表达人TLR2和TLR6的NF - κB - SEAP报告基因HEK293细胞中分泌的碱性磷酸酶（SEAP），PamCySOH显示出显著活性，尽管不如PamCysSK4或PamCysSK。因此，PamCysOH作为一种形成囊泡的TLR2/6激动剂和免疫反应刺激剂尤其令人关注。
