Liposome leakage and increased cellular permeability induced by guanidine-based oligomers: effects of liposome composition on liposome leakage and human lung epithelial barrier permeability

Abstract

Over the decades, guanidine-based oligomer groups have been one of the most widely used antimicrobial agents. Reportedly, these cationic oligomers cause serious damage to microorganisms but have low toxicity to humans. However, public concerns regarding the guanidine group have rapidly grown after the fatal misuse of these oligomers as humidifier disinfectants, which resulted in thousands of fatalities in South Korea. Herein, we investigated liposome leakage and cellular permeability changes caused by polyhexamethylene guanidine (PHMG) and polyhexamethylene biguanide (PHMB), both representative guanidine-based oligomers. The leakage of zwitterionic liposomes, induced by cationic oligomers, was more extensive than that of negative liposomes, indicating that oligomer adsorption onto lipid head groups via electrostatic interaction cannot fully explain the induced lipid membrane damage. Furthermore, lipid packing parameters, including intrinsic curvature, cholesterol content, and lipid phases, affected liposome leakage, particularly for PHMG. Cellular permeability tests were performed using an A549 cell monolayer model and a respiratory 3D tissue model, revealing that PHMG and PHMB damaged cell membranes and reduced cell barrier function. Furthermore, liposome leakage induced by PHMG and PHMB was higher in human lung surfactant-mimicking liposomes than that observed in Escherichia coli-mimicking liposomes. These results indicated that human cells are susceptible to guanidine-based oligomers. Considering that the interaction of oligomers and cell membranes is a major mechanism of toxicity initiation, this study provides crucial insights into the action of these disinfectants on mammalian cells.

Fig. 1. Liposome leakage test of encapsulated 5(6)-carboxyfluorescein from (a) DOPC (zwitterionic) and (b) DOPG (negative) liposomes with the selective additions of 23 mg L⁻¹ of PHMG or PHMB and TX-100 (0.5 M). PHMG, polyhexamethylene guanidine; PHMB, polyhexamethylene biguanide. 5(6)-Carboxyfluorescein leakage of the (c) DOPC and (d) DOPG liposomes with 23 mg L⁻¹ of PHMG and PHMB.

Fig. 2. 5(6)-Carboxyfluorescein leakage from DOPC liposomes by (a) PHMG and (b) PHMB at concentrations ranging from 1.2 mg L⁻¹ to 46 mg L⁻¹, and the leakage of the DOPG liposomes by (c) PHMG and (d) PHMB with the same concentration ranges. PHMG, polyhexamethylene guanidine; PHMB, polyhexamethylene biguanide.

Fig. 3. Alterations in (a) DOPC and (b) DOPG liposome sizes after 30 min incubating with different concentrations of PHMG and PHMB. In (b), the y-axis representing liposome size has a break ranging from 160 nm to 185 nm. (c) Image obtained after incubation of DOPG and DOPC liposomes with 462 mg L⁻¹ PHMB and PHMG solution for 30 min. Precipitation can be observed when DOPG liposomes were incubated with 462 mg L⁻¹ of PHMB and PHMG. PHMG, polyhexamethylene guanidine; PHMB, polyhexamethylene biguanide.

Fig. 4. Diagrammatic illustration of the effects of lipid head charges on the possible liposome leakage caused by guanidine-based disinfectants. Liposome leakage mechanism of (a) DOPC and (b) DOPG liposomes. PHMG, polyhexamethylene guanidine.

Fig. 5. Effects of DOPE contents in (a) DOPC and (b) POPC liposomes on the 5(6)-carboxyfluorescein leakage caused by PHMG. PHMG, polyhexamethylene guanidine.

Fig. 6. Effects of cholesterol contents in (a) DOPC and (b) POPC liposomes on the 5(6)-carboxyfluorescein leakage. f denotes the mole fraction of cholesterol in DOPC or POPC liposomes caused by PHMG. PHMG, polyhexamethylene guanidine.

Fig. 7. (a) Liposome leakage test for 5(6)-carboxyfluorescein encapsulating DOPC and DPPC liposomes by 23 mg L⁻¹ of PHMG. (b) 5(6)-Carboxyfluorescein leakage of the DOPC and DPPC liposomes with PHMG. PHMG, polyhexamethylene guanidine.

Fig. 8. Changes in airway barrier permeability by PHMG or PHMB. The (a) A549 cell monolayer model and (b) respiratory 3D tissue model were exposed to 23 mg L⁻¹ of PHMG or PHMB for 1, 3, 6, 12 and 24 h. The permeability of the airway barrier was assessed by measuring the FITC-dextran cellular flux. Results are provided as a percentage for vehicle control (vc). Additionally, 1% Triton X-100 was used as a positive control (pc). Values significantly different from vehicle control: **p < 0.01. PHMG, polyhexamethylene guanidine; PHMB, polyhexamethylene biguanide.

Fig. 9. 5(6)-Carboxyfluorescein leakage of the DOPC, E. coli mimic liposomes, and lung surfactant mimic liposomes induced by (a) PHMG and (b) PHMB. The E. coli mimic liposomes consist of 80% DOPE and 20% DOPG, and lung surfactant mimic liposomes consist of 70% DPPC, 10% DOPC, 10% DOPG, and 10% DOPE. PHMG, polyhexamethylene guanidine; PHMB, polyhexamethylene biguanide.