In vivo treatment of Propionibacterium acnes infection with liposomal lauric acids

Abstract

Propionibacterium acnes (P. acnes) is a Gram-positive bacterium strongly associated with acne infection. While many antimicrobial agents have been used in clinic to treat acne infection by targeting P. acnes, these existing anti-acne agents usually produce considerable side effects. Herein, the development and evaluation of liposomal lauric acids (LipoLA) is reported as a new, effective and safe therapeutic agent for the treatment of acne infection. By incorporating lauric acids into the lipid bilayer of liposomes, it is observed that the resulting LipoLA readily fuse with bacterial membranes, causing effective killing of P. acnes by disrupting bacterial membrane structures. Using a mouse ear model, we demonstrated that the bactericidal property of LipoLA against P. acne is well preserved at physiological conditions. Topically applying LipoLA in a gel form onto the infectious sites leads to eradication of P. acnes bacteria in vivo. Further skin toxicity studies show that LipoLA does not induce acute toxicity to normal mouse skin, while benzoyl peroxide and salicylic acid, the two most popular over-the-counter acne medications, generate moderate to severe skin irritation within 24 h. These results suggest that LipoLA hold a high therapeutic potential for the treatment of acne infection and other P. acnes related diseases.

Keywords: Propionibacterium acnes; antimicrobial delivery; bacterial infection; free fatty acid; nanoparticle.

Figure 1

Schematics of using liposomal lauric acids (LipoLA) to treat acne infection caused by Propionibacterium acnes (P. acnes) bacteria. (A) Anatomic illustration of skin with inflammatory acne. (B) Structure and composition of LipoLA consisting of phospholipid, cholesterol and lauric acid. (C) Hypothesized mechanism of action; LipoLA fusing into bacterial membranes. (D) Anatomic illustration of skin after LipoLA treatment. (E) Hydrodynamic size (diameter, nm) and surface charge (zeta potential, mV) of the prepared LipoLA.

Figure 2

FRET measurements of the fusion between LipoLA and P. acnes bacteria. LipoLA were labeled with both a fluorescent donor (C₆NBD) and a fluorescent acceptor (DMPE-RhB) at a proper molar ratio that the acceptor maximally quenched the fluorescence emission from the donor. The FRET-pair labeled LipoLA were then incubated with P. acnes at various bacterial concentrations. After removing the excess LipoLA, all samples were excited at 470 nm and the fluorescence emission spectra were recorded (Inset). A rise in emission intensity of C₆NBD at 520 nm was observed, indicating the occurrence of spatial separation of C₆NBD and DMPE-RhB due to the fusion between LipoLA and bacterial membranes.

Figure 3

In vitro antimicrobial activity of LipoLA against P. acnes and morphology of P. acnes after LipoLA treatment. LipoLA were incubated with P. acnes for 5 h under anaerobic condition. (A) P. acnes bacteria were diluted and spotted on agar plates, and the bacterial number was quantified. LipoLA completely killed the bacteria. PBS was used as a negative control. Data represents mean ± SD of three independent experiments. UD: undetectable. (B) Following LipoLA or PBS treatment, the bacteria were fixed and imaged by scanning electron microscope (SEM). A destruction of bacterial membranes and an absence of fimbriae were observed with the LipoLA treated sample (right panel) as compared to the PBS treated sample (left panel).

Figure 4

In vivo antimicrobial activity of LipoLA against P. acnes through intradermal injection. Different concentrations of LipoLA (2, 4, 6, or 8 mg/mL) were mixed with P. acnes and immediately injected into mouse ears (n=6 per group), followed with bacteria quantification at 24 h. BPO (16 mg/mL) was used as a positive control. PBS and none P. acnes inoculation were served as negative controls. Data represents mean ± SD of three independent experiments. UD: undetectable.

Figure 5

In vivo therapeutic efficacy of LipoLA for the treatment of P. acnes infection through topical administration. P. acnes (10⁶ CFU) were inoculated onto a wound generated on a mouse ear (n=6 per group), followed with topical application of LipoLA gel daily for 2 days successively. At 72 h, the ear was collected, and the remaining amount of P. acnes was quantified. Commercial BPO cream from a drug store was used as a positive control. PBS gel and none P. acnes inoculation were served as negative controls. Data represents mean ± SD of three independent experiments. UD: undetectable.

Figure 6

Toxicity study of LipoLA on mouse back skin. LipoLA gel was topically applied onto shaved mouse back skin (n=6 per group). After 24 h, the gel was removed and the skin was analyzed. Blank PBS gel was used as a negative control. BPO, salicylic acid (SA), and Nair^® cream were used as positive controls. (A) Morphology of the skin after treatment imaged by a camera. (B) Draize’s irritation scores indicating erythema. (C) Edema scores of the skin. (D) Back skin was cross-sectioned, stained with hematoxylin and eosin (H&E), and viewed by a microscope. Data is representative of three separate experiments with similar results.