Title：Understanding Experimentally Compatible Bactericidal Activity of Dicationic Ionic Liquids: A Mechanistic Insight into the Effect of Functional Groups by MD Simulations

Journal: The Journal of Physical Chemistry B 

Original link：https://doi.org/10.1021/acs.jpcb.5c00942

Reporter：Fujun Yang-25-master

       Dicationic ionic liquids (DCILs) show a promising innovative potential as antibacterial agents to help overcome the antibiotic-resistant bacteria crisis worldwide. Changing ionic head groups, side chain lengthening, functionalizing, and modifying the hydrophobic/hydrophilic character of the IL structure influence their interaction strength with the bacterial cell wall. Nevertheless, deep molecular-level insights are a prerequisite in fully realizing the antibacterial mechanism of DCILs with varied functionalities and structures. Here, we selected three DCILs based on the recently investigated bis-imidazolium dibromide family, DCIL-1, DCIL-3, and DCIL-5, with the functional groups 2-hydroxybutyl, 2-hydroxy-3-(methacryloyloxy)propyl, and 2-hydroxy-3-phenoxypropyl, respectively. Current all-atom molecular dynamics (MD) simulations and free-energy calculations consistency with our earlier experimental assays confirmed the order of (DCIL-5 > DCIL-1 > DCIL-3) for their bactericidal activity against Escherichia coli (E. coli). The dication insertion is the dominant driving force for the bacterial bilayer disruption and rupture. The MD results revealed that the antibacterial activity of bulky DCILs was due to the interplay between the electrostatic and hydrophobic interactions. It further disclosed the antibacterial mechanism consisting of the dication adsorption on the bacterial membrane lipids through electrostatic attraction, the flip motion of dications for finding suitable orientation in close vicinity to the lipid bilayer’s surface, key hydrogen-bond forming simultaneously with the lipid’s head groups to promote the penetration of the adjacent hydrophobic group to the lipid bilayer center. The penetration process could increase the average surface area per lipid, decrease the lipid tail ordering and the bilayer thickness, and improve the lipid lateral diffusion and bilayer fluidity, resulting in lipid bilayer rupture and bacterial membrane lysis. The strongest antibacterial activity was demonstrated by DCIL-5, which had a 2-hydroxyl-3-phenoxypropyl functional group and a high relative hydrophobicity and lipophilicity that allowed it to permeate the bacterial cell walls efficiently. This research sheds light on the microscopic interactions between DCILs having various functional groups and Gram-negative bacterial membranes, providing crucial insights for screening and the rational design of new cationic agents as efficient antibacterial materials.

     The global incidence of bacterial infections is on the rise. In particular, Gram-negative bacteria, due to their complex cell membrane structures, exhibit stronger adaptability and resistance to conventional antibiotics, which have become a major threat to the fields of medical care and public health. Novel antibacterial materials represent a core breakthrough for addressing antibiotic resistance. Therefore, redesigning the structures of existing antibiotics and exploring new medicinal materials with enhanced antibacterial properties have become imperative approaches.

     Existing experiments have confirmed that dicationic ionic liquids (DCILs) possess high-efficiency antibacterial activity against both Gram-positive and Gram-negative bacteria, with relatively low cytotoxicity. However, research on their molecular mechanisms remains unclear. Specifically, there is a lack of clarity on how functional groups regulate the microscopic interactions between DCILs and bacterial membranes to influence antibacterial efficacy. Furthermore, molecular-level analyses are insufficient, including the dynamic process of DCIL insertion into bacterial membranes, the quantitative impact on membrane structures, and changes in free energy. This knowledge gap fails to provide a theoretical basis for redesigning existing antibiotic structures and exploring new medicinal materials with enhanced antibacterial performance.

     Thus, all-atom molecular dynamics simulations and free energy calculations were selected to elucidate the regulatory mechanisms of different functional groups on the interactions between DCILs and Escherichia coli membranes.

 1. Structural formula of the three target antibacterial DCILs from the bis-imidazolium dibromide family with some key atomic labels.

2. Structures of three phospholipids forming the ternary bilayer membrane model of E. coli (90 POPE, 24 POPG, and 6 CL) in the MD simulations.

 3. Final snapshots of the simulation boxes after 200 ns with approximate box dimensions in the z and x directions. The dication adsorption and insertion into the lipid bilayer are seen in the snapshots. The dications are shown with the VDW model, the P atoms in the lipid headgroup in the tan sphere, and the fatty acid hydrocarbon chains in cyan

4. (a) Comparison of the number density profiles, ρ(z), along the direction perpendicular to the lipid bilayer surface, z, for four selected carbon

atoms of different dications. The center of the bilayer membrane is located at z = 0. (b) Comparison of the ρ(z) of three carbon atoms of the sn2 chain

of POPE with the key tail carbon atoms of the three dication-containing systems. (c) Computed minimum distances during the 200 ns simulation

period between the head nitrogen or the POPE lipid’s tail carbon atoms and the tail carbon atoms of both sides of each dication. The structural formula

of the DCILs, the POPE molecule, and some atomic labels are embedded on top of the graphs

5. Computed average number density profiles for the Phosphorus atom of the lipid, Ow atom of water, and Br− along the z direction from the

simulations of three considered DCILs in the hydrated bilayer systems

6. Comparison of partial radial distribution functions, g(r), for three DCIL-containing systems between key atomic sites of dications (C1, C3,

C12, OH) and the lipid bilayer’s POPE atomic sites (P, OL) in the left and middle graphs. The (P−P) and some other pair structural correlations in the

right-hand panels, including the lipid oxygen or the dication hydroxyl oxygen with the water oxygen or Br− are compared

7. Comparison of the structural correlations between the head phosphorus (P) atom of three phospholipid types of the E. coli membrane model

and the key ring’s carbon (C3 and C12) atoms of the three bis-imidazolium dications

 8. (a) Evaluation of the absolute values of the mutual intermolecular (Coulombic and van der Waals) interactions between each dication and

water or POPE/POPG/CL lipids. (b) The average number of H-bonds between the dication and water or POPE/POPG/CL lipids. (c) Deuterium

order parameters, SCD, for the sn1 (oleoyl chain) and sn2 (palmitoyl chain) tails of the hydrated POPE lipid bilayer in contact with three DCILs.

Carbon 1 refers to the carbon connected to the polar headgroup, and the numbering order of carbon atoms increases toward the tail methyl group

 9. Computed lateral MSD along the x−y plane for the head phosphorus atom of the lipid membrane from the last 10,000 ps of simulation in the presence of aqueous DCILs

 10. Free-energy profile of dication insertion from the bulk water to the lipid bilayer center using the COM dication−lipid center distance (ξ) as the reaction coordinate from the umbrella sampling approach.

    Through all-atom molecular dynamics (MD) simulations and free energy calculations, it is revealed that the insertion of dicationic cations is the primary driving force for the disruption and rupture of bacterial lipid bilayers.

    The results of molecular dynamics simulations demonstrate that the antibacterial activity of dicationic ionic liquids (DCILs) with larger molecular volumes stems from the synergistic effect of electrostatic interactions and hydrophobic interactions. The study further elucidates the underlying antibacterial mechanism: dicationic cations are adsorbed onto bacterial membrane lipids via electrostatic attraction, followed by a flipping motion to adopt an optimal orientation near the surface of the lipid bilayer. Meanwhile, they form key hydrogen bonds with lipid headgroups, thereby facilitating the penetration of adjacent hydrophobic groups into the core of the lipid bilayer. This penetration process increases the average surface area per lipid molecule, reduces the orderliness of lipid tails and the thickness of the lipid bilayer, enhances the lateral diffusion capacity of lipids and the fluidity of the bilayer membrane, and ultimately leads to the rupture of the lipid bilayer and the dissolution of the bacterial membrane.

    This study clarifies the microscopic interactions between dicationic ionic liquids with different functional groups and the cell membranes of Gram-negative bacteria, providing important insights for the screening and rational design of novel cationic antibacterial agents as high-efficiency antibacterial materials.