Cationic Liposomes with Different Lipid Ratios: Antibacterial Activity, Antibacterial Mechanism, and Cytotoxicity Evaluations

Abstract

Due to their strong bacterial binding and bacterial toxicity, cationic liposomes have been utilized as effective antibacterial materials in many studies. However, few researchers have systematically compared their antibacterial activity with their mammalian cell cytotoxicity or have deeply explored their antibacterial and cytotoxicity mechanisms. Here, we prepared a series of cationic liposomes (termed CLs) using dimethyldioctadecylammonium chloride (DODAC) and lecithin at different molar ratios. CLs have the ability to effectively bind with Gram-positive and Gram-negative bacteria through electrostatic and hydrophobic interactions. Further, the CLs with high molar ratios of DODAC (30 and 40 mol%) can disrupt the bacterial wall/membrane, efficiently inducing the production of reactive oxygen species (ROS). More importantly, we carefully compared the antibacterial activity and the mammalian cell cytotoxicity of various CLs differing in DODAC contents and liposomal concentrations and revealed that, whether they are bacterial or mammalian cells, an increasing DODAC content in CLs can lead to an elevated cytotoxicity level. Further, there exists a critical DODAC contents (>20 mol%) in CLs to endow them with effective antibacterial ability. However, the variation in the DODAC content and liposomal concentration of CLs has different degrees of influence on the antibacterial activity or cytotoxicity. For example, CLs at high DODAC content (i.e., CL0.3 and CL0.4) could effectively kill both types of bacterial cells but only cause negligible toxicity to mammalian cells. We believe that a systematic comparison between the antibacterial activity and the cytotoxicity of CLs with different DODAC contents will provide an important reference for the potential clinical applications of cationic liposomes.

Keywords: antimicrobial; bacteria; biocompatibility; cationic liposome; cytotoxicity.

Scheme 1

Scheme showing the structures of diverse CLs differing in various DODAC/lecithin molar ratios and the comparison between their antibacterial activity and cytotoxicity. CL0, CL0.1, CL0.2, CL0.3, and CL0.4 in the scheme represent the CLs with a DODAC content of 0, 10, 20, 30, and 40 mol%, respectively.

Figure 1

Characterization of CLs. (a) TEM image of CL0.4 and corresponding size distribution histogram (inset). Scale bar = 50 nm. (b) Hydrodynamic diameter distributions of CL0, CL0.1, CL0.2, CL0.3, and CL0.4 measured by DLS. (c) Changes of the hydrodynamic diameters of different CLs within 3 weeks. (d) Zeta potentials of CLs in PBS.

Figure 2

Antibacterial activities of CLs. Line charts showing viable bacterial percentages of S. aureus (a) and E. coli (b) after being treated with CL0, CL0.1, CL0.2, CL0.3, or CL0.4 at various concentrations for 5 h.

Figure 3

(a) SEM images of S. aureus and E. coli cells before and after the treatment of CL0.4 for 5 h. Scale bar = 2 μm. (b) Zeta potentials of S. aureus and E. coli bacteria after treatment with PBS or different CLs for 5 min in PBS.

Figure 4

(a) Relative ROS levels in S. aureus and E. coli bacteria after incubation with various CLs for 5 h. (b) Agarose gel electrophoresis results of the extracted DNA from S. aureus (left panel) or E. coli (right panel) cells treated with PBS (control) or various CLs. M: DNA Ladder DL10000. 0, 1, 2, 3, 4, and 5 represent S. aureus and E. coli treated with PBS (control), CL0, CL0.1, CL0.2, CL0.3, CL0.4, and CL0.5, respectively.

Figure 5

Relative viabilities of NIH 3T3 cells and HPAEpiCs after incubation with different CLs for 5 and 24 h. (a) NIH 3T3 cells, 5 h, (b) HPAEpiCs, 5 h, (c) NIH 3T3 cells, 24 h, (d) HPAEpiCs, 24 h.

Figure 6

Flow cytometry analysis results of NIH 3T3 cells after treatment with the culture medium (control) or various concentrations of CLs (400 μg/mL) for 5 h. Before analysis, the cells were stained with annexin V-FITC and PI.