Directional Supramolecular Assembly of π-Amphiphiles with Tunable Surface Functionality and Impact on the Antimicrobial Activity

Abstract

This article elucidates H-bonding-regulated directional supramolecular assembly of naphthalene diimide (NDI)-derived unsymmetric cationic bola-shaped π-amphiphiles and systematic investigations on the thermodynamics of their interaction with bacteria mimic lipid vesicles and antimicrobial activity with mechanistic insights. Four NDI-amphiphiles (NDI-1, NDI-2, NDI-3, and NDI-2a) have been studied, all of which contain a central NDI chromophore, a nonionic wedge, an amine containing a head group, and a hydrazide group. In NDI-2 and NDI-2a, the hydrophilic wedge and the head group (pyridine) are the same but the location of the hydrazide group is different. On the basis of this difference, the pyridyl groups are displayed at the outer and inner walls of the vesicle, respectively. Isothermal titration calorimetry (ITC) studies revealed the spontaneous interaction of NDI-2 assembly with bacteria membrane mimic DPPE liposome (ΔG = −6.35 kcal/mol), whereas the NDI-2a assembly did not interact at all, confirming a strong influence of the H-bonding-regulated functional group display. On the other hand, the location of the hydrazide group remains the same in NDI-1, NDI-2, and NDI-3, but they differ in the head group structure. ITC binding studies confirmed spontaneous interaction of all three assemblies with DPPE liposome with negative ΔG values following the order NDI-1 > NDI-2 > NDI-3, indicating significant influence of the structure of the head group on the interaction with the model membrane. In fact, in all cases, the interaction was favorable both by enthalpy and entropy contribution, indicating dual involvement of the electrostatic interaction and hydrophobic effect. Notably, ΔS value for NDI-1 containing a tertiary amine head group was found to be significantly higher than that for NDI-3 containing a primary amine, which is attributed to the enhanced hydrophobic effect in the former case. Furthermore, ITC experiments revealed no interaction by any of these assemblies with the mammalian cell membrane mimic liposome, indicating their high selectivity toward bacterial membranes. Antimicrobial activity studies showed NDI-2 to be lethal selectively against Gram-positive bacteria, whereas NDI-2a did not show any activity. NDI-3 with a primary amine showed moderate activity but no selectivity over the erythrocytes. NDI-1 with the tertiary amine group was found to be the most outstanding candidate, exhibiting broad-spectrum antimicrobial activity with very low minimum inhibitory concentration values of 15.8 and 62 μg/mL for Staphylococcus aureus and Escherichia coli, respectively, and high selectivity over erythrocytes. These results fully corroborate with the physical insights obtained from the ITC studies on their interaction with the model liposome. Control molecules, lacking either the NDI chromophore or the hydrazide nonionic containing wedge, did not exhibit any notable antibacterial activity. Live-dead assay with fluorescence microscopy studies indicated that the antimicrobial activity of NDI-1 operates through the membrane disruption pathway similar to that of the host defense peptides.