Department of Chemical Engineering, Worcester Polytechnic Institute, Worcester, MA 01609, USA.
Department of Biomedical Engineering, Worcester Polytechnic Institute, Worcester, MA 01609, USA.
Colloids Surf B Biointerfaces. 2018 Jul 1;167:229-238. doi: 10.1016/j.colsurfb.2018.04.016. Epub 2018 Apr 5.
The human antimicrobial peptide LL37 is promising as an alternative to antibiotics due to its biophysical interactions with charged bacterial lipids. However, its clinical potential is limited due to its interactions with zwitterionic mammalian lipids leading to cytotoxicity. Mechanistic insight into the LL37 interactions with mammalian lipids may enable rational design of less toxic LL37-based therapeutics. To this end, we studied concentration- and time-dependent interactions of LL37 with zwitterionic model phosphatidylcholine (PC) bilayers with quartz crystal microbalance with dissipation (QCM-D). LL37 mass adsorption and PC bilayer viscoelasticity changes were monitored by measuring changes in frequency (Δf) and dissipation (ΔD), respectively. The Voigt-Kelvin viscoelastic model was applied to Δf and ΔD to study changes in bilayer thickness and density with LL37 concentration. At low concentrations (0.10-1.00 μM), LL37 adsorbed onto bilayers in a concentration-dependent manner. Further analyses of Δf, ΔD and thickness revealed that peptide saturation on the bilayers was a threshold for interactions observed above 2.00 μM, interactions that were rapid, multi-step, and reached equilibrium in a concentration- and time-dependent manner. Based on these data, we proposed a model of stable transmembrane pore formation at 2.00-10.0 μM, or transition from a primarily lipid to a primarily protein film with a transmembrane pore formation intermediate state at concentrations of LL37 > 10 μM. The concentration-dependent interactions between LL37 and PC bilayers correlated with the observed concentration-dependent biological activities of LL37 (antimicrobial, immunomodulatory and non-cytotoxic at 0.1-1.0 μM, hemolytic and some cytotoxicity at 2.0-13 μM and cytotoxic at >13 μM).
人抗菌肽 LL37 因其与带电荷的细菌脂质的物理相互作用而有望替代抗生素。然而，由于其与两性离子哺乳动物脂质的相互作用导致细胞毒性，其临床潜力受到限制。对 LL37 与哺乳动物脂质相互作用的机制深入了解可能能够合理设计毒性更低的基于 LL37 的治疗方法。为此，我们使用石英晶体微天平（QCM-D）研究了 LL37 与两性离子模型磷脂酰胆碱（PC）双层的浓度和时间依赖性相互作用。通过测量频率（Δf）和耗散（ΔD）的变化，分别监测 LL37 质量吸附和 PC 双层粘弹性变化。应用 Voigt-Kelvin 粘弹性模型研究了 LL37 浓度变化对双层厚度和密度的影响。在低浓度（0.10-1.00 μM）下，LL37 以浓度依赖的方式吸附在双层上。对Δf、ΔD 和厚度的进一步分析表明，肽在双层上的饱和是观察到高于 2.00 μM 的相互作用的一个阈值，这些相互作用是快速的、多步骤的，并且以浓度和时间依赖的方式达到平衡。基于这些数据，我们提出了一个模型，即在 2.00-10.0 μM 时形成稳定的跨膜孔，或者在浓度高于 10 μM 时，从主要是脂质到主要是蛋白质膜的转变，中间状态是形成跨膜孔。LL37 与 PC 双层的浓度依赖性相互作用与观察到的 LL37 浓度依赖性生物活性（在 0.1-1.0 μM 时具有抗菌、免疫调节和非细胞毒性，在 2.0-13 μM 时具有溶血和一些细胞毒性，在>13 μM 时具有细胞毒性）相关。
