Effects of lipid membrane composition on the distribution of biocidal guanidine oligomer with solid supported lipid membranes

Abstract

Polyhexamethylene guanidine (PHMG) is a cationic antimicrobial oligomer that has been used prevalently over the past few decades. However, due to the lack of inhalation toxicity assessment of PHMG, it has caused severe health damage, including fatal lung fibrosis, after being used as one of the major active ingredients of humidifier disinfectants in Korea. Because the first step of the entry of PHMG into airway is its association with cell membranes, the distribution of PHMG between lipid membranes and water is very important to know the depositional flux in the respiratory systems and related toxic mechanisms. We developed a quantitative method to determine the distribution constant (K _lipw) of PHMG between solid supported lipid membranes and water and evaluated the effects of lipid membrane compositions on the K _lipw of PHMG. PHMG accumulated into anionic lipid membranes rapidly compared to into cationic or zwitterionic lipid membranes, suggesting fast adsorption of PHMG onto anionic lipid head groups. K _lipw values with anionic/zwitterionic lipid mixtures were higher than K _lipw values with anionic lipids only, potentially due to the later phase separation after preferential interaction between PHMG and anionic lipids in lipid mixtures. In addition, K _lipw values increased with increasing single acyl chain lipid content in unsaturated lipids and decreasing cholesterol content. These results imply that changes in lipid spontaneous curvature and lipid bilayer packing density also affect the membrane distribution of PHMG.

Fig. 1. Rates of PHMG distribution with solid supported lipid membranes with different head group charges. DOTAP, DOPC, and DOPG have positive, zwitterion, and negative head group charges, respectively, with the same acyl chain length (C18:1). C_w is the free concentration in water after settling solid supported lipid membranes and C₀ is the initial PHMG concentration (C₀ = 4.04 mg L⁻¹). The error bars represent standard deviations of triplicate samples.

Fig. 2. Concentration dependence on the (a) lipid accumulation and (b) lipid–water distribution coefficient (K_lipw) of PHMG with 16:1 PC. C₀ is the initial concentration and each point represents the mean value of duplicate analyses.

Fig. 3. (a) Lipid accumulation rates of PHMG (C₀ = 4.04 mg L⁻¹) using three different lipid membranes with different head group charges. Dotted lines indicate that the Langmuir adsorption model fits using eqn (3). (b) The lipid–water distribution constant (K_lipw) of PHMG between water and three lipid membranes calculated after 240 h of incubation. Error bars represent standard deviations of triplicates.

Fig. 4. Diagrammatic illustration of the possible distribution mechanism of PHMG into solid supported lipid membranes with (a) DOPG (anionic lipid), (b) DOPC (zwitterionic lipid), and (c) DOTAP (cationic lipid).

Fig. 5. (a) Lipid–water distribution constant (K_lipw) of PHMG (C₀ = 5.0 mg L⁻¹) between water and lipid membranes with different compositions after 72 h of incubation. POPC has a zwitterion head group, and POPG and DOPS are anionic lipid membranes. POPC/POPG and POPC/DOPS are zwitterionic/anionic lipid mixtures prepared at a 1 : 1 molar ratio. Error bars represent the standard deviations of three replicates. (b) Illustration showing the lipid lateral separation caused by binding PHMG to the lipid membrane composed with zwitterionic/anionic lipid mixtures. Strong interactions between PHMG and anionic lipid membranes promote the clustering of anionic lipid membranes around PHMG, which causes lipid rearrangement/segmentation. This figure was inspired by Teixeira et al.

Fig. 6. Effects of lyso PG content on the distribution constants (K_lipw) of PHMG (C₀ = 4.0 mg L⁻¹) and (a) DOPG (18:1/18:1 PG) and (b) POPG (16:0/18:1 PG) lipid membranes. K_lipw values were obtained after 72 h of incubation. Error bars indicate the standard deviations of three replicate analyses.

Fig. 7. The effects of cholesterol content (mole fraction of cholesterol, f is in the range of 0 and 0.65) on the distribution constant (K_lipw) of PHMG (C₀ = 4.0 mg L⁻¹ and 6.0 mg L⁻¹) and 16:1 PC lipid membranes. K_lipw values were obtained after 72 h of incubation. Error bars indicate the standard deviations of three replicate analyses.